WO2008001903A1 - Fad-conjugated glucose dehydrogenase gene - Google Patents

Fad-conjugated glucose dehydrogenase gene Download PDF

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Publication number
WO2008001903A1
WO2008001903A1 PCT/JP2007/063147 JP2007063147W WO2008001903A1 WO 2008001903 A1 WO2008001903 A1 WO 2008001903A1 JP 2007063147 W JP2007063147 W JP 2007063147W WO 2008001903 A1 WO2008001903 A1 WO 2008001903A1
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WIPO (PCT)
Prior art keywords
fad
amino acid
polynucleotide
acid sequence
glucose dehydrogenase
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PCT/JP2007/063147
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French (fr)
Japanese (ja)
Inventor
Takako Yada
Koji Miyamoto
Michinari Honda
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Ikeda Food Research Co., Ltd.
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=38845665&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2008001903(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Ikeda Food Research Co., Ltd. filed Critical Ikeda Food Research Co., Ltd.
Priority to US12/866,071 priority Critical patent/US8492130B2/en
Priority to JP2008522658A priority patent/JP4665235B2/en
Publication of WO2008001903A1 publication Critical patent/WO2008001903A1/en
Priority to US13/920,445 priority patent/US8882978B2/en
Priority to US14/510,076 priority patent/US9340816B2/en
Priority to US15/135,375 priority patent/US9663811B2/en
Priority to US15/496,935 priority patent/US9976125B2/en
Priority to US15/955,650 priority patent/US20180237754A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
    • C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/301—Reference electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/302—Electrodes, e.g. test electrodes; Half-cells pH sensitive, e.g. quinhydron, antimony or hydrogen electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T29/00—Metal working
    • Y10T29/49—Method of mechanical manufacture
    • Y10T29/49002—Electrical device making
    • Y10T29/49117—Conductor or circuit manufacturing

Definitions

  • the present invention relates to a novel gene (polynucleotide) encoding a flavin adenine dinucleotide (FAD) -binding glucose dehydrogenase, a method for producing the enzyme using a transformed cell recombined with the gene,
  • the present invention relates to a FAD-bound glucose dehydrogenase, and a method for measuring gnolecose, a glucose measuring reagent composition, a biosensor for measuring dalcose, etc. characterized by using the enzyme.
  • the amount of blood gnolecose is an important marker of diabetes. Diabetes testing includes clinical tests in hospital laboratories, as well as simple measurements (Point-of-Care Testing: POCT) such as simple tests by medical staff and self-tests by patients themselves.
  • POCT Point-of-Care Testing
  • This simple measurement is performed by a measuring device (POCT device) such as a gno-les course diagnostic kit or a biosensor.
  • a measuring device such as a gno-les course diagnostic kit or a biosensor.
  • glucose oxidase has been used in these POCT devices.
  • glucose oxidase is affected by the dissolved oxygen concentration and an error occurs in the measured value, it is recommended to use gnolecose dehydrogenase that is not affected by oxygen.
  • the glucose dehydrogenase includes nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a coenzyme.
  • NAD nicotinamide adenine dinucleotide
  • NADP nicotinamide adenine dinucleotide phosphate
  • coenzyme-linked glucose dehydrogenases that use quinone (PQQ), flavin adenine dinucleotide (FAD), etc. as coenzymes.
  • PQQ quinone
  • FAD flavin adenine dinucleotide
  • coenzyme-linked glucose dehydrogenase is less susceptible to contamination components than non-coenzyme-linked glucose dehydrogenase, has high measurement sensitivity, and in principle, makes the POCT device inexpensive. It has the advantage that it can be manufactured.
  • the conventional PQQ-linked glucose dehydrogenase has the disadvantage that it is poor in stability and also reacts with manoleose and galactose.
  • Maltose is a sugar used for infusion, and when PQQ-linked glucose dehydrogenase reacts with maltose.
  • Blood glucose The POCT device displays blood glucose levels higher than actual. For this reason, as a result of unnecessary insulin injections by patients, hypoglycemia accidents such as disturbance of consciousness and falling into a coma have occurred, which is a major problem.
  • the present blood glucose POCT apparatus is used for the purpose of simply measuring blood sugar, and is becoming increasingly important as a means of patient self-management and treatment. Since the spread of measuring devices (Self-Monitoring of Blood Glucose: SMBG) to homes is steadily expanding, the demand for measurement accuracy is considered to be very high.
  • SMBG Blood Glucose
  • Patent Document 1 a novel soluble coenzyme-linked glucose dehydrogenase using FAD as a coenzyme from Aspergillus terreus.
  • Patent Document 1 This coenzyme-linked glucose dehydrogenase of Patent Document 1 oxidizes the hydroxyl group at the 1-position of dalcose, has excellent substrate recognition for glucose, is not affected by dissolved oxygen, and has low strength against maltose.
  • vs. Gunorekosu activity versus maltose activity as a 100 Q / o is 5. / 0 or less, versus galactose activity than 5%) are those having excellent properties not in until now referred.
  • Patent Document 1 the coenzyme-linked glucose dehydrogenase of Patent Document 1 is isolated and extracted from a liquid culture of a wild microorganism (for example, Aspergillus microorganism), The production was limited. In addition, the amount of enzyme produced is extremely small, and a large amount of sugar is bound to the enzyme, which is covered with a different type of sugar than the N-type or O-type sugar chains that are bound to normal enzymes.
  • a wild microorganism for example, Aspergillus microorganism
  • Patent Document 1 Pamphlet of International Publication No. 2004/058958
  • Patent Document 2 JP 59-25700 A
  • Non-patent document 1 Biochem. Biophys. Acta., 139,277-293, 1967
  • coli which is generally used as a recombinant host, and is limited to host microorganisms (such as Pseudomonas) that produce PQQ. As a result, there is a problem that a recombinant must be made.
  • the present invention solves the above-mentioned problems, and has excellent properties such as excellent reactivity to glucose, heat stability, and substrate recognition property, and low force and low activity on maltose.
  • Novel gene polynucleotide
  • the object is to provide a method, a glucose measuring reagent composition, a biosensor for measuring gnolecose, and the like.
  • the present inventor encodes the gene to express FAD-bound gnolecose dehydrogenase significantly in Aspergillus oryzae strains.
  • AVPWV amino acid sequence
  • XI and X2 are aliphatic amino acids, X3 and X6 are branched amino acids, and X4 and X5 are polycyclic amino acids or aromatic amino acids).
  • Polynucleotide (XI and X2 are aliphatic amino acids, X3 and X6 are branched amino acids, and X4 and X5 are polycyclic amino acids or aromatic amino acids).
  • polypeptide having an FAD-linked glucose dehydrogenase activity consisting of an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence of amino acid sequence (a), or
  • a polypeptide comprising an amino acid sequence having 70% or more homology with the amino acid sequence (a) and having FAD-bound glucose dehydrogenase activity.
  • Amino acid sequence Sense primer comprising nucleotide sequence that encodes AGVPWV and FAD-bound glucose dehydrogenation derived from Aspergillus oryzae Reverse primer consisting of the nucleotide sequence at the 3 'end of the polynucleotide encoding the enzyme, or an amino acid sequence: antisense primer against the nucleotide sequence encoding AGVPWV and Aspergillus oryzae (5k AD binding)
  • Amino acid sequence A polynucleotide that hybridizes under stringent conditions with a probe having a nucleotide sequence that encodes AGVPWV and encodes a polypeptide having FAD-linked glucose dehydrogenase activity.
  • FA characterized in that FAD-conjugated glucose dehydrogenase having an action of dehydrogenating glucose is collected from the culture obtained by culturing the above transformed cells.
  • a reagent composition for measuring dalcose which contains the above-mentioned FAD-linked glucose dehydrogenase.
  • a biosensor for measuring dalcose characterized by using the FAD-bound glucose dehydrogenase described above.
  • a FAD-bound gnolecose dehydrogenase having excellent substrate recognizability to glucose, excellent strength, and low activity against maltose is obtained.
  • the ability to produce homogeneous and large-scale products using genetic recombination technology is S.
  • the enzyme produced in this way can control the amount of sugar, which has been a problem with FAD-bound glucose dehydrogenase, according to the purpose, so by preparing an enzyme with reduced sugar content, In blood glucose measurement, etc., it is possible to change the action on sugars (such as glucose) in the sample.
  • FIG. 1 shows a calibration curve of glucose concentration with an enzyme-immobilized electrode.
  • FIG. 2 Shows the detection result of the target gene by PCR.
  • M 200 bp DNA ladder marker (manufactured by Takara Bio Inc.) 1: Aspergillus oryzae NBRC42682: Aspergillus oryzae NBRC53753: Aspergillus oryzae NBRC62154: Aspergillus oryzae NBRC41815: Aspergillus * oryzae NBRC42206: Aspergillus NBCRC
  • FIG. 3 Shows the detection result of the target gene by Southern hybridization. The symbols in the figure are the same as in Figure 2.
  • the amino acid sequence is: X, X2-X3-X 4-X5-X6
  • XI and X2 are the same or different aliphatic amino acids, X3 and X6 are the same or different branched amino acids, and X4 and X5 are the same or different heterocyclic amino acids or aromatic amino acids), It is one of the technically important points that a polypeptide consisting of 6 amino acids is contained, and for this reason, the enzyme is significantly expressed in the microbial cells. The expressed enzyme does not necessarily need to be secreted outside the cell. It may stay. In contrast, as specifically shown in the examples of the present specification, even a gene encoding an enzyme considered to be FAD-linked glucose dehydrogenase from the homology of the entire amino acid sequence, etc. The gene encodes a polypeptide consisting of a sequence, and the gene does not express a protein having FAD-linked glucose dehydrogenase activity.
  • the six amino acid sequences are preferably located at positions 202 to 207 of a polypeptide that is a FAD-linked glucose dehydrogenase, or at least one force XI of X1 to X6 is alanine ( A), X2 is glycine (G), X3 is valine (V), X4 is proline (P), X5 is tryptophan (W), or X6 is valine (V).
  • A X2 is glycine
  • V valine
  • X4 proline
  • X5 tryptophan
  • W tryptophan
  • X6 valine
  • the amino acid sequence AGVPWV SEQ ID NO: 4
  • SEQ ID NO: 4 can be cited as a preferred example.
  • FAD-linked glucose dehydrogenase refers to catalyzing a reaction of dehydrogenating (oxidizing) the hydroxyl group at the 1-position of glucose in the presence of an electron acceptor, and thereby acting on glucose.
  • it means a soluble protein having an action on maltose of 10% or less, and the enzyme is characterized by the following properties.
  • Flavin adenine dinucleotide as a coenzyme
  • oxygen is not an electron acceptor
  • those having the amino acid sequence AGVPWV are particularly preferably those derived from Aspergillus oryzae.
  • Examples of such strains include NBRC5375, NBR C4079, NBRC4203, NBRC4214, NBRC4268, NBRC5238, NBRC6215, NBRC30104, and NBRC30113 as shown in Table 1 below. I can do it.
  • Amino acid sequence: AGVPWV is the vicinity of 202-207 (from NBRC5375 strain) when the starting amino acid M of the signal sequence portion is the first in the amino acid sequence of the enzyme (in the case of enzymes derived from other strains). Is included in the location corresponding to the position).
  • amino acid sequence of the FAD-linked gnolecose dehydrogenase expressed by Aspergillus oryzae NBRC5375 is SEQ ID NO: 1 (including signal peptide)
  • the nucleotide sequence of the chromosomal DNA encoding it is SEQ ID NO: 2
  • the cDNA corresponding to the amino acid shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, respectively.
  • the base sequence encoding the amino acid sequence AGVPWV is GCTGGTGTTCCATGGGT T (SEQ ID NO: 5).
  • polynucleotide of the present invention encodes the following polypeptide (a), (b) or (c) in addition to the above-mentioned one derived from the strain of Aspergillus oryzae.
  • polypeptide comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in amino acid sequence (a) and having FAD-linked glucose dehydrogenase activity, or
  • polynucleotide of the present invention includes the following polynucleotide (d), (e) or (f):
  • nucleotides (e) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence (d) under stringent conditions and encodes a polypeptide having FAD-linked glucose dehydrogenase activity.
  • the polypeptide of (b) or (c) above comprises the amino acid sequence: X1-X2-X3-X4-X5-X6, or the polynucleotide of (e) or (f) Those containing a base sequence encoding the amino acid sequence are preferred. Furthermore, it is preferable that this amino acid sequence is AGVPWV.
  • an amino acid sequence or a base sequence having 70% or more homology shows at least 70% identity over the entire length of the reference sequence to be compared, and preferably 75%. % Or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more.
  • sequence identity percentages are published or commercially available with algorithms that compare a reference sequence as a query sequence. Can be calculated using software. For example, BLAST, FASTA, or GENETYX (manufactured by Software Development Co., Ltd.) can be used, and these can be used with default parameters.
  • hybridization under stringent conditions for hybridizing between polynucleotides are, for example, 50. /. Honolemamide, 5 X SSC (150 mM sodium chloride, 15 mM trisodium citrate, 10 mM sodium phosphate, ImM ethylenediamine tetraacetic acid, ⁇ 7.2), 5 ⁇ Denhardt's solution, 0.1% SDS, 10 Exemplify washing the filter at 42 ° C in 0.2 X SSC after 42 ° C incubation with% dextran sulfate and 100 zg / mL denatured salmon sperm DNA.
  • the polynucleotide of the present invention includes a sense primer comprising a base sequence encoding amino acid sequence: AGVPWV, and a polynucleotide encoding a FAD-bound gnolecose dehydrogenase derived from Aspergillus oryzae.
  • a polynucleotide encoding a polypeptide having FAD-linked glucose dehydrogenase activity which has a DNA fragment that can be amplified by PCR using a combination of forward primers consisting of a base primer having a 5'-end base sequence.
  • the polynucleotide of the present invention hybridizes with a probe comprising the nucleotide sequence encoding the amino acid sequence: AGVPWV under stringent conditions and encodes a polypeptide having FAD-bound glucose dehydrogenase activity. Polynucleotides.
  • the base sequence encoding the amino acid sequence AGVPWV is (GCTGGTGTTCCATG GGTT).
  • various conditions relating to the above-mentioned PCR and hybridization under stringent conditions can be appropriately selected by those skilled in the art according to the description of the examples in the present specification.
  • the polynucleotide of the present invention has an enzyme activity value for maltose of 10% or less, preferably 5% or less, more preferably, when the enzyme activity value for D-gnolecose is 100%.
  • the enzyme activity value for D-galactose is 5% or less, preferably 3% or less, preferably
  • specific activity per protein is measured, for example, in a state where the culture supernatant is concentrated and confirmed as a single band by SDS-PAGE, as described in Example 7 of the present specification. It has been done.
  • polynucleotide means a nucleoside phosphate ester (ATP (adenosine triphosphate), GTP (guanosine triphosphate) in which purine or pyrimidine is bonded to a sugar by / 3-N-daricoside.
  • ATP adenosine triphosphate
  • GTP guanosine triphosphate
  • Phosphate CTP (cytidine triphosphate), UTP (uridine triphosphate); or dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate) ), DTTP (deoxythymidine triphosphate)) is a molecule that binds more than 100 molecules.
  • chromosomal DNA that encodes FAD-linked gnolecose dehydrogenase, mRNA transcribed from chromosomal DNA, and mRNA. It includes cDNA and polynucleotides that have been PCR-amplified using them as saddles.
  • Olionucleotide refers to a molecule in which 2-99 nucleotides are linked.
  • Polypeptide means a molecule composed of 30 or more amino acid residues linked to each other by amide bonds (peptide bonds) or unnatural residue linkages. Includes those that have been added or those that have been artificially chemically modified.
  • polynucleotide (gene) of the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
  • a polynucleotide that is a chromosomal DNA represented by SEQ ID NO: 2 is prepared, for example, aspergillus oryzae NBRC5375 strain, a chromosomal DNA library, and the FAD-binding type derived from Aspergillus terreus described in Patent Document 1.
  • DOGAN Database of the Genomes Analyze
  • the labeling of the probe is preferably performed by any method known to those skilled in the art, for example, a force non-RI method that can be performed by a radioisotope (RI) method or a non-RI method.
  • RI radioisotope
  • non-RI method examples include a fluorescence labeling method, a piotine labeling method, a chemiluminescence method, and the like, but it is preferable to use a fluorescence labeling method.
  • Fluorescent substances can be selected from those that can bind to the base moiety of the oligonucleotide as appropriate.
  • Cyanine dyes eg, Cy DyeTM M series Cy3, Cy5, etc.
  • rhodamine 6G reagent N-acetoxy- N2 -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), etc.
  • AAF N-acetoxy- N2 -acetylaminofluorene
  • AAIF iodine derivative of AAF
  • the polynucleotide represented by SEQ ID NO: 3 was prepared as described above using a cDNA library as a saddle shape, as specifically described in the examples of the present specification, for example. It can also be obtained by various PCR methods known to those skilled in the art using a set of oligonucleotide primers (probes), or by RT-PCR using total RNA or mRNA extracted from Aspergillus oryzae NBRC5375 strain as a saddle type.
  • the size of the primer should be 15-40 bases, preferably 15-30 bases, considering that it will satisfy the specific annealing with the vertical DNA. It is.
  • the annealing temperature depends on Tm (melting temperature), in order to obtain highly specific PCR products, select primers that are close to each other with a Tm value of 55-65 ° C.
  • oligonucleotide probe or oligonucleotide primer set can be prepared, for example, by cleaving cDNA, which is a polynucleotide of the present invention, with an appropriate restriction enzyme.
  • the polynucleotide of the present invention is modified, for example, from the above-mentioned FAD-linked glucose dehydrogenase cDNA derived from Aspergillus oryzae NBRC5375 strain by a known mutation introduction method or mutagenesis PCR method. Can be created. Furthermore, it must be obtained from the chromosomal DNA of Aspergillus oryzae strains other than the NBRC5375 strain or its cDNA library by the probe hybridization method using the oligonucleotide prepared based on the nucleotide sequence information of SEQ ID NO: 1. Can do. In hybridization, the polynucleotide can be obtained by variously changing the stringent conditions.
  • Stringent conditions are defined by the concentration of salt in the hybridization and washing process, the concentration of organic solvent (formaldehyde, etc.), temperature conditions, etc., for example, as described in US Pat. No. 6,100,037, etc. Various conditions known to those skilled in the art, such as those disclosed, can be employed.
  • the recombinant vector of the present invention is a cloning vector or an expression vector, and an appropriate one is used according to the type of polynucleotide as an insert, the purpose of use, and the like.
  • expression vectors for in vitro transcription prokaryotic cells such as Escherichia coli and Bacillus subtilis, filamentous fungi such as yeast and mold, Expression vectors suitable for eukaryotic cells such as insect cells and mammalian cells can also be used.
  • Examples of the transformed cell of the present invention include prokaryotic cells such as Escherichia coli and Bacillus subtilis, and yeast. , Eukaryotic cells such as molds, insect cells, mammalian cells and the like can be used. These transformed cells can be prepared by introducing a recombinant vector into cells by a known method such as electroporation, calcium phosphate method, ribosome method, DEAE dextran method. Specific examples of the recombinant vector and the transformed cell include the recombinant vector shown in the Examples below, and transformed Escherichia coli and transformed mold using the vector.
  • the FAD-linked gnolecose dehydrogenase of the present invention is produced by expressing DNA in a microorganism such as Escherichia coli, the origin, promoter, ribosome binding site, DNA cloning site capable of replicating in the microorganism, If an expression vector is prepared by recombining the above-mentioned polynucleotide into an expression vector having a terminator sequence, etc., host cells are transformed with this expression vector, and the resulting transformant is cultured.
  • Type glucose dehydrogenase can be mass-produced with microorganisms.
  • an FAD-linked glucose dehydrogenase fragment containing the arbitrary region can be obtained.
  • it can be expressed as a fusion protein with another protein.
  • the target FAD-bound glucose dehydrogenase can also be obtained by cleaving this fusion protein with an appropriate protease.
  • the expression vector for E. coli include pUC system, pBluescriptll, pET expression system, pGEX expression system, and pCold expression system.
  • the polynucleotide is a eukaryotic cell having a promoter, a splicing region, a poly (A) addition site, etc.
  • FAD-linked glucose dehydrogenase can be produced in eukaryotic cells by creating a recombinant vector by inserting it into an expression vector for use and introducing it into a eukaryotic cell. It can be maintained in a cell like a plasmid, or it can be maintained in a chromosome.
  • expression vectors examples include pKAl, pCDM8, pSVK3, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYE82.
  • pIND / V5-His, pFLAG-CMV_2, pEGFP_Nl, pEGFP-Cl, etc. are used as expression vectors, FAD-linked glucose dehydrogenase poly- hydrase can be used as a fusion protein to which various tags such as His tag, FLAG tag, and GFP are added.
  • Eukaryotic cells are commonly used for cultured mammalian cells such as monkey kidney cell COS_7, Chinese hamster ovary cell CH 2 O, budding yeast, fission yeast, mold, silkworm cell, Xenopus egg cell, etc. FAD-coupled glucose dehydrogenation Any eukaryotic cell may be used as long as it can express the enzyme.
  • known methods such as electroporation, calcium phosphate method, ribosome method, DEAE dextran method can be used.
  • Jung is preferred.
  • the target After expressing FAD-conjugated glucose dehydrogenase in prokaryotic cells or eukaryotic cells, the target is obtained from a culture (a culture solution or a medium composition containing an enzyme secreted outside the cell).
  • a culture a culture solution or a medium composition containing an enzyme secreted outside the cell.
  • known separation operations can be combined.
  • treatment with denaturing agents and surfactants such as urea, heat treatment, pH treatment, ultrasonic treatment, enzyme digestion, salting out solvent precipitation method, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-P AGE , Isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, affinity chromatography (tag sequence-based methods and polyclonal antibodies specific to FAD coenzyme-linked glucose dehydrogenase) Antibody, and a method using a monoclonal antibody).
  • denaturing agents and surfactants such as urea, heat treatment, pH treatment, ultrasonic treatment, enzyme digestion, salting out solvent precipitation method, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-P AGE , Isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, affinity chromatography (tag sequence-based methods and polyclonal antibodies specific to FAD coenzyme
  • FAD-linked glucose dehydrogenase can be obtained by a recombinant DNA technique using the polynucleotide (cDNA or translation region thereof) of the present invention.
  • cDNA or translation region thereof polynucleotide of the present invention.
  • the polynucleotide is recombined into an appropriate expression vector by a known method, the polynucleotide is encoded in prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, mold, insect cells and mammalian cells. It is possible to express a large amount of the FAD-linked gnolecose dehydrogenase. It is also possible to introduce a polynucleotide optimized for force codon usage, which is the same amino acid sequence, according to the host. The key to sugar chains
  • a recombinant vector is prepared by inserting the polynucleotide described above into a vector having a promoter to which RNA polymerase can bind.
  • FAD-linked glucose dehydrogenase can be produced in vitro by adding the vector to an in vitro translation system such as a rabbit reticulocyte lysate or wheat germ extract containing RNA polymerase corresponding to the promoter.
  • promoters to which RNA polymerase can bind include T3, T7 and SP6.
  • the vector containing these promoters include pKAl, pCDM8, pT3 / ⁇ 718, ⁇ 7 / 319, and pBluescriptll.
  • the recombinant FAD-linked gnolecose dehydrogenase of the present invention can be produced by the method described above.
  • a FAD-bound type of gonorlecose dehydrogenase is an enzyme that catalyzes the reaction of dehydrogenating gnolecose in the presence of an electron acceptor, and is not particularly limited as long as the change due to this reaction can be used.
  • substances that can be used in the medical field and clinical field such as measuring and measuring reagents for glucose in samples containing biological materials, as well as reagents for erasing, and using coenzyme-linked gnorolecose dehydrogenase It can also be used in production.
  • the reagent composition for glucose measurement of the present invention may be mixed together to form a single reagent. When there are components that interfere with each other, the components are separated so that they are combined appropriately. May be. These may be prepared as a solution or powdery reagent, and may be prepared as a test paper or an analytical film by containing them in a suitable support such as filter paper or film. A standard reagent containing a deproteinizing agent such as perchloric acid or a quantitative amount of gnolecose may be attached. The amount of enzyme in the composition is preferably about 0.1 to 50 units per sample. Examples of the specimen for quantifying gnolecose include plasma, serum, spinal fluid, saliva, urine and the like.
  • the biosensor of the present invention is a glucose sensor that is used in a reaction layer containing the FAD-bound glucose dehydrogenase of the present invention as an enzyme and measures the glucose concentration in a sample solution.
  • an electrode system consisting of a working electrode, its counter electrode, and a reference electrode is formed on an insulating substrate using a method such as screen printing, and is in contact with the hydrophilic polymer and oxidized with this electrode system. It is produced by forming an enzyme reaction layer containing a reductase and an electron acceptor.
  • a sample solution containing a substrate is dropped onto the enzyme reaction layer of this biosensor, the enzyme reaction layer is dissolved and the enzyme reacts with the substrate, and the electron acceptor is reduced accordingly.
  • the reduced electron acceptor is electrochemically oxidized.
  • the nanosensor can measure the substrate concentration in the sample solution from the obtained oxidation current value.
  • Chemical substances that excel in electron transfer are chemical substances that are generally called ⁇ electron mediators '', ⁇ mediators '', or ⁇ redox mediators ''.
  • the electron mediators and redox mediators listed in Special Table 2002-526759 may be used. Specific examples include an osmium compound, a quinone compound, and a fluoric compound.
  • the enzyme is preferably diluted appropriately so that the final concentration is 0.:! To 1. Ounit / mL.
  • the enzyme activity unit (unit) of the enzyme is an enzyme activity that oxidizes 1 ⁇ mol of glucose per minute.
  • the enzyme activity of the FAD-linked gnolecose dehydrogenase of the present invention can be measured by the following method.
  • 0.1 M potassium phosphate buffer ( ⁇ 7.0) 1. OmL, 1. OM D—Gnolecose 1. OmL, 3 mM 2,6-Dichlorophenol indophenol (DCIP) 0 ⁇ 14 mL, 3 mM 1—Methoxy 5—Methylphenmethyl methyl sulfate 0.2 mL, 0.61 mL of water is added to a 3 mL quartz cell (optical path length lcm), set in a spectrophotometer equipped with a thermostatic cell holder, and 5 ° C at 37 ° C. After incubation for 5 minutes, add 0.05 mL of the enzyme solution, and then measure the change in absorbance (A ABSZmin) at 600 ⁇ m of DCIP.
  • a ABSZmin absorbance
  • the enzyme activity that reduces 1 ⁇ mol of DCIP per minute is substantially equivalent to the enzyme activity limit.
  • the enzyme activity was determined from the change in absorbance according to the following formula.
  • the enzyme is preferably diluted as appropriate so that the final concentration is preferably 0.2 to 0.9 mg / mL.
  • the protein concentration in the present invention was determined using the Bio-Rad Protein Assay, a protein concentration measurement kit that can be purchased from Japan Bio's Rad Co., Ltd., and according to the instruction manual, bovine serum albumin (BSA, Wako Pure Chemical Industries, Ltd.). It can be obtained by converting the calibration curve force created as a reference material.
  • BSA bovine serum albumin
  • RT-PCR was performed under the following conditions, and assumed to be about 1.8 kbp FAD-conjugated glucose dehydrogenase. A PCR product containing the gene was obtained.
  • Primer 1 5 '-tgggatcctatgctcttctcactggcat-3' ( ⁇ ⁇ ⁇ lj number 6)
  • Reaction conditions Reverse transcription reaction 42 ° C, 30 minutes (1 cycle)
  • Denaturation 94 C, 30 seconds, annealing 45 ° C, 30 seconds, extension reaction 72.
  • C 1 minute 30 seconds (25 cyclores)
  • the PCR amplification fragment obtained in (4) was cleaved with restriction enzymes BamHI and Hindlll, and DNA Ligation Kit Ver.2.1 (Takara Bio) was used for the PUC18 vector (Takara Bio) treated with the same restriction enzymes. And a plasmid containing a gene presumed to be FAD-linked glucose dehydrogenase was prepared.
  • the plasmid obtained in (5) was introduced into E. coli JM109 Competent Cell (Takara Bio Inc.) for transformation.
  • LB plate containing ampicillin sodium manufactured by Wako Pure Chemical Industries, Ltd.
  • directly PCR is estimated to be FAD-conjugated gnorecose dehydrogenase
  • transformants were obtained using ampicillin sodium-containing LB plates.
  • Example 1 (1) Of the wet cells obtained in Example 1 (1), 0.25 g was frozen with liquid nitrogen, ground, and chromosomal DNA was extracted by a conventional method.
  • a vector capable of expressing this gene was prepared by binding a gene presumed to be a FAD-linked gnolecose dehydrogenase amplified using the above.
  • Transformation is basically carried out according to the method described in known literature 2 and publicly known literature 3 (gene manipulation technology for koji mold for sake, Katsuya Gomi, Shukyo, P494-502, 2000). Acquired.
  • Aspergillus oryzae NS4 strain was used as the host to be used. This strain is bred in a brewery laboratory in 1997 as described in known literature 1, and is used for analysis of transcription factors, breeding of high-producing strains of various enzymes, etc. Is available
  • an improved amylase-based promoter derived from Aspergillus oryzae described in publicly known document 2 was used, and the chromosomal DNA obtained in (2) was used as a cage in the lower part.
  • a vector capable of expressing this gene by binding the gene (AO090005000449 gene) presumed to be FAD-linked glucose dehydrogenase amplified using the primers used in 2 (SEQ ID NO: 8 and SEQ ID NO: 9) Prepared.
  • Transformation was basically carried out according to the methods described in known literature 2 and known literature 3 to obtain transformants.
  • the 604th force and the 606th ATG and the retinal sequence at that time are the GCTGGTGTTCCATGGGTT sequence shown in SEQ ID NO: 5 in the gene presumed to be FAD-bound glucose dehydrogenase derived from Aspergillus oryzae NBRC 5375 strain, The other sequences were perfectly matched.
  • the translated amino acid sequence is shown in SEQ ID NO: 1 and compared in the same manner.
  • the starting amino acid M of AO09000 5000449 is the first
  • the 202nd M is the FAD-bound dalcose derived from Aspergillus oryzae NBRC 5375 strain
  • the amino acid sequence encoded by the gene deduced to be dehydrogenase was the sequence AGVPWV shown in SEQ ID NO: 4, and the other sequences were completely identical.
  • SEQ ID NO: 2 The result of sequencing the gene presumed to be FAD-bound glucose dehydrogenase derived from Aspergillus olisee NBRC 5375 strain in the recombinant strain obtained in Example 2 is shown in SEQ ID NO: 2.
  • the translated amino acid sequence is shown in SEQ ID NO: 1 and compared in the same manner.
  • the starting amino acid M of AO09000 5000449 preumed to be choline dehydrogenase
  • the 202nd M is the Aspergillus oryzae NBRC5375.
  • the amino acid sequence encoded by the gene presumed to be a strain-derived FAD-linked glucose dehydrogenase was AGVPW V shown in SEQ ID NO: 4, and the other sequences were identical.
  • the strains of Examples 1 and 2 and the strains of Comparative Examples have similar gene sequences in the gene presumed to be FAD-linked glucose dehydrogenase.
  • the gene sequence derived from Aspergillus oryzae NBRC5375 Compared with the IO IJ gene of AO090005000449 derived from the strain, the arrangement of ATG at the 656th force and the 658th is the sequence GCTGGTGTTCCATGGGTT shown in SEQ ID NO: 5, and compared with the amino acid sequence.
  • the amino acid M near the 202nd position was found to be AGVPWV shown in SEQ ID NO: 4.
  • DNA was extracted from the wet cells cultured based on the strains obtained in Example 2 and Comparative Example by a conventional method, and a part of the gene assumed to be the FAD-bound glucose dehydrogenase was probed as a Southern. Detection was performed by blotting.
  • DNA fragments containing the gene assumed to be FAD-linked glucose dehydrogenase linked to the improved promoter of amylase derived from Aspergillus oryzae contain the same number of copies. It has been found.
  • Example 2 that is, it was found that the strains obtained in Example 2 and the Comparative Example each contained the same copy number of the gene by transformation.
  • RNA was extracted from the wet cells cultured based on the strains obtained in Example 2 and the comparative example by a conventional method, and a part of the gene assumed to be the FAD-binding glucose dehydrogenase was probed as a northern region. Detection was performed by blotting.
  • the transformed strain was estimated to be the same as that of the FAD-bound gnolecose dehydrogenase gene bound to the improved amylase promoter derived from Aspergillus oryzae. Fragments were detected.
  • the strains obtained in Example 2 and Comparative Example could be judged to have transcribed the gene assumed to be the present FAD-binding glucose dehydrogenase to the same degree of RNA.
  • the culture supernatant was 53 U / mL. Although the FAD-bound glucose dehydrogenase activity was confirmed, no activity was confirmed in the culture supernatant and the cell-free extract.
  • Example 1 (1) For several other strains of Aspergillus oryzae, in the same manner as in Example 1 (1) (2), FAD-linked glucose dehydrogenation in their culture supernatant and cell-free extract (CFE) Elementary activity was confirmed.
  • CFE cell-free extract
  • chromosome DNA was extracted in the same manner as in Example 2- (1), and the sequence of the approximately 1.9 kbp fragment amplified using the primers described in SEQ ID NOs: 6 and 7 was determined. Comparison was made with the sequence of number 2 and the chromosomal DNA sequence of AO090005000449. Further, the translated amino acid sequence was compared with the sequence shown in SEQ ID NO: 1 and the amino acid sequence 1J of AO090005000449. These results are shown in Table 1 below together with the results of Examples 1 to 3 and Comparative Example. Regarding the sequence, the presence or absence of the amino acid sequence AGVPWV described in Example 3- (1) is shown in Table 1.
  • the chromosomal DNA sequence derived from Aspergillus oryzae NBRC4203 differed from the sequence of SEQ ID NO: 2 in 4 bases (135C ⁇ A, 437G ⁇ A, 532G ⁇ A, 1263C ⁇ T). Furthermore, the amino acid sequence obtained by translating the chromosomal DNA sequence derived from Aspergillus oryzae NBRC4203 differed from the sequence of SEQ ID NO: 1 by 2 amino acids (129V ⁇ I, 386A ⁇ V).
  • chromosomal DNA sequence derived from Aspergillus oryzae NBRC30104 was different in the arrangement 1J of SEQ ID NO: 2 and 4 bases (135C ⁇ A, 413C ⁇ A, 437G ⁇ A, 532G ⁇ A). Furthermore, translated amino acid sequence of chromosomal DNA derived from Aspergillus oryzae NBRC30104 The sequence was different from the sequence of SEQ ID NO: 1 by 2 amino acids (121R ⁇ S, 129V ⁇ I). It was speculated that these amino acid sequence differences did not directly affect the expression of FAD-bound glucose dehydrogenase.
  • chromosomal DNA sequences of Aspergillus oryzae NBRC4181 and 4220 were completely consistent with the chromosomal DNA IJ of AO090005000449.
  • the gene presumed to be FAD-bound glucose dehydrogenase derived from Aspergillus oryzae NBRC5375 is a gene encoding an active FAD-bound glucose dehydrogenase.
  • the gene presumed to be FAD-linked gonorlecose dehydrogenase derived from Aspergillus oryzae NBRC100959 was not a gene encoding active FAD-linked glucose dehydrogenase. .
  • the AO090005000449 gene encodes an amino acid sequence that is very similar to the amino acid sequence of the FAD-bound gnolecose dehydrogenase derived from the NBRC5375 strain. It is thought that. However, unexpectedly, the present inventor found for the first time that the enzyme is not expressed in the sequences of the AO090005000449 gene, the NBRC4181 gene, and the 4220 gene as shown in the comparison column. It was. Using the same expression system, the presence or absence of the expression of FAD-bound glucose dehydrogenase is caused only by the difference in the above-mentioned sequences.
  • AGVP WV Amino acid sequence contained in type dulcose dehydrogenase: AGVP WV appears to be an important sequence for adopting the higher-order structure of FAD-linked glucose dehydrogenase. If AGVPWV is lacking, it is expressed by causing endoplasmic reticulum stress. It is inferred that protein degradation and / or suppression of expression occurs. It is important for functional expression that the amino acid sequence: AGVPWV exists near the 202nd position when the starting amino acid M is the first position.
  • the culture supernatant of the cells of Example 2 obtained in Example 5 was concentrated with Vivacel 2 (manufactured by Viva Science) with a molecular weight cut-off of 10,000, and then replaced with distilled water. 323 UZmg of purified enzyme was obtained. Enzymes derived from other strains that showed FAD-linked glucose dehydrogenase activity could be purified in the same manner. When these purified enzymes were subjected to SDS-PAGE, a single band of about 86 kDa was confirmed. For this enzyme, the operability, substrate specificity and coenzyme were examined. The enzyme activity was measured according to the enzyme activity measurement method described above.
  • the purified enzyme was reacted with 500 mM D-glucose in the presence of 8.66 mM DCIP, and the reaction product was quantified with the D-Dalconic acid / D-Dalcono ⁇ -Lataton quantification kit.
  • the FAD-bound glucose dehydrating enzyme of the present invention is an enzyme that catalyzes the reaction of oxidizing the hydroxyl group at the 1-position of D-gnolecose. It was.
  • the enzyme activity of the purified enzyme was measured according to the enzyme activity measurement method, using D-glucose, maleoleose, and D-galactose as the substrate for the activity measurement reaction solution in the enzyme activity measurement method described above.
  • the activity value of the enzyme for D-gnolecose was 100%
  • the enzyme activity value for maltose was 2.1%
  • the enzyme activity value for D-galactose was 0.99%.
  • the GC electrode was connected to a potentiostat BAS100BZW (manufactured by BAS), the solution was stirred at 37 ° C., and +500 mV was printed on the silver salt / silver reference electrode.
  • a 1M D-glucose solution was added to these systems to a final concentration of 5, 10, 20, 30, 40, and 50 mM, and a steady-state current value was measured for each addition.
  • this current value was plotted against the known gnolecose concentration (5, 10, 20, 30, 40, 50 mM), a calibration curve was created (Fig. 1). From this, it was shown that quantorecose can be quantified with an enzyme-immobilized electrode using the FAD-linked glucose dehydrogenase of the present invention.
  • Glucose Nacalai 1% (W / V), defatted soybean (Showa Sangyo) 2% (W / V), Cone steep liquor (SUNEI KAGAKU) 0.5% (W / V ), Magnesium sulfate heptahydrate (Nacalaine) 0.1% (WZV) and a liquid medium consisting of water are adjusted to pH 6.0, 10 mL is placed in a thick test tube, and autoclaved at 121 ° C for 20 minutes. did.
  • Example 4 in this cooled liquid medium, Aspergillus oryzae NBRC4268 strain, NBRC5375 strain, NBRC6215 strain, which have been confirmed to have glucose dehydrogenase activity in the culture broth, and the culture broth Aspergillus oryzae NBRC4181 strain, NBRC4220 strain, and NBRC100959 strain, in which this enzyme activity is not observed, were inoculated into each test tube, cultured at 30 ° C for 43 hours with shaking, and then each was treated with a wet centrifuge using a centrifuge. The body was recovered.
  • the wet cells obtained in (1) were frozen in liquid nitrogen, crushed, and chromosomal DNA was extracted by a conventional method.
  • Primers synthesized based on the sequence of SEQ ID NO: 2 using each DNA extracted in (2) as a template PCR was carried out using 1 and 3 under the following conditions to obtain a PCR product containing an approximately 1.9 kbp FAD-linked glucose dehydrogenase gene.
  • Primer 3 5 '-ttatgctcttctcactggcattcctgagtgccctgt-3' ( ⁇ ⁇ ⁇ lj number 10)
  • PCR was performed under the following conditions using primer 3 and primer 5 synthesized based on the amino acid sequence: AGVPWV.
  • Primer 3 5 '-ttatgctcttctcactggcattcctgagtgccctgt-3, ( ⁇ ⁇ ⁇ IJ number 10) Phuima 1 5 : 5 -aacccatggaacaccagc-3 ′ ( ⁇ self column number 12)
  • FIG. 2 shows the results of detection of the target gene by PCR. Only the polynucleotide encoding the FAD-linked glucose dehydrogenase derived from Aspergillus oryzae having glucose dehydrogenase activity in the culture medium was confirmed to have the amplification of the size expected by force PCR. In addition, even when PCR is carried out using the DNA obtained in (2) directly as a template, the FAD-linked gnorolecose dehydrogenase derived from Aspergillus oryzae having glucose dehydrogenase activity in the culture medium is similarly encoded. Only those polynucleotides that were able to confirm the amplification of the size expected by PCR.
  • Probe: 5 (FITC) -gctggtgttccatgggtt-3 '(SEQ ID NO: 5).
  • Fig. 3 shows the results of detection of the target gene by Southern hybridization. It can be seen that only the polynucleotide encoding the FAD-linked gnolecose dehydrogenase derived from Aspergillus oryzae having glucose dehydrogenase in the culture medium can be detected by Southern hybridization. For confirmation by Southern hybridization, the PCR product obtained in (9) of Example 9 was placed on a nylon membrane (Hybond_N +, manufactured by GE Healthcare). The same result can be obtained even if it is fixed to the above.
  • Example 9 It was confirmed by the method shown in Example 9 and / or Example 10 that the polynucleotide was able to be confirmed as a polynucleotide encoding an FAD-linked gnolecose dehydrogenase derived from Aspergillus oryzae that secretes and produces glucose dehydrogenase in the culture medium.
  • the gene was secreted and produced in large quantities in the culture supernatant.
  • Table 2 shows the results of calculating the average activity (3 strains) per culture medium for each mutagenized recombinant (presumed to be a single copy). These results strongly suggest that these 6 amino acids, particularly the 205th to 207th amino acids, are important for the expression of the activity of Aspergillus * olise FAD-linked gnolecose dehydrogenase.
  • the FAD-conjugated gnolecose dehydrogenase encoded by the polynucleotide of the present invention does not substantially act on maltose in the measurement of blood glucose, it can be used for a more accurate self blood glucose measurement (SMBG) apparatus. It can greatly contribute to the self-management and treatment of diabetic patients.
  • SMBG self blood glucose measurement

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Abstract

Disclosed are: a novel gene (polynucleotide) encoding an FAD-conjugated glucose dehydrogenase having excellent properties such as excellent reactivity to glucose, excellent thermal stability and excellent substrate-recognition performance and also having low reactivity to maltose; a process for the production of the enzyme by using a transformant cell recombined with the gene; a method for the determination of glucose, a reagent composition for use in the determination of glucose, a biosensor for use in the determination of glucose and others, each using the enzyme. Specifically, disclosed are: a polynucleotide encoding an FAD-conjugated glucose dehydrogenase, comprising a polypeptide having the following amino acid sequence: X1-X2-X3-X4-X5-X6 [wherein X1 and X2 independently represent an aliphatic amino acid residue; X3 and X6 independently represent a branched amino acid residue; and X4 and X5 independently represent a heterocyclic amino acid residue or an aromatic amino acid residue]; and others.

Description

明 細 書  Specification
FAD結合型グルコース脱水素酵素遺伝子  FAD-linked glucose dehydrogenase gene
技術分野  Technical field
[0001] 本発明は、フラビンアデニンジヌクレオチド (FAD)結合型グルコース脱水素酵素を コードする新規な遺伝子（ポリヌクレオチド）、該遺伝子により組換えられた形質転換 細胞を用いる該酵素の製造方法、組換え FAD結合型グルコース脱水素酵素、並び に、該酵素を使用することを特徴とするグノレコースの測定方法、グルコース測定試薬 組成物、及びダルコース測定用のバイオセンサ等に関する。  [0001] The present invention relates to a novel gene (polynucleotide) encoding a flavin adenine dinucleotide (FAD) -binding glucose dehydrogenase, a method for producing the enzyme using a transformed cell recombined with the gene, The present invention relates to a FAD-bound glucose dehydrogenase, and a method for measuring gnolecose, a glucose measuring reagent composition, a biosensor for measuring dalcose, etc. characterized by using the enzyme.
背景技術  Background art
[0002] 血中グノレコース量は糖尿病の重要なマーカーである。糖尿病の検査は、病院検査 室等での臨床検査の他、診療スタッフ等による簡易検査や患者自身による自己検査 といった簡易測定（Point-of-Care Testing:POCT)が実施されている。  [0002] The amount of blood gnolecose is an important marker of diabetes. Diabetes testing includes clinical tests in hospital laboratories, as well as simple measurements (Point-of-Care Testing: POCT) such as simple tests by medical staff and self-tests by patients themselves.
[0003] この簡易測定は、グノレコース診断キットやバイオセンサ等の測定装置 (POCT装置 )によって実施されている力 従来、これらの POCT装置にはグルコース酸化酵素が 用レ、られてきた。しかし、グルコース酸化酵素は溶存酸素濃度の影響を受け、計測値 に誤差が生じるため、酸素の影響を受けないグノレコース脱水素酵素の使用が推奨さ れている。  [0003] This simple measurement is performed by a measuring device (POCT device) such as a gno-les course diagnostic kit or a biosensor. Conventionally, glucose oxidase has been used in these POCT devices. However, since glucose oxidase is affected by the dissolved oxygen concentration and an error occurs in the measured value, it is recommended to use gnolecose dehydrogenase that is not affected by oxygen.
[0004] グルコース脱水素酵素には、ニコチンアミドアデニンジヌクレオチド（NAD)又はニコ チンアミドアデニンジヌクレオチドリン酸 (NADP)を補酵素とする補酵素非結合型グ ルコース脱水素酵素と、ピロ口キノリンキノン (PQQ)、フラビンアデニンジヌクレオチド (FAD)等を補酵素とする補酵素結合型グルコース脱水素酵素がある。その中で補 酵素結合型グルコース脱水素酵素は、補酵素非結合型グルコース脱水素酵素に比 べ夾雑成分の影響を受けにくいこと、測定感度が高いこと、更に原理上、 POCT装 置を安価に製造することが可能であるとレ、う利点を有してレ、る。  [0004] The glucose dehydrogenase includes nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a coenzyme. There are coenzyme-linked glucose dehydrogenases that use quinone (PQQ), flavin adenine dinucleotide (FAD), etc. as coenzymes. Among them, coenzyme-linked glucose dehydrogenase is less susceptible to contamination components than non-coenzyme-linked glucose dehydrogenase, has high measurement sensitivity, and in principle, makes the POCT device inexpensive. It has the advantage that it can be manufactured.
[0005] し力しながら、従来の PQQ結合型グルコース脱水素酵素は安定性が低ぐしかもマ ノレトースゃガラタトースにも反応してしまうという欠点を有している。マルトースは輸液 に用いられる糖であり、 PQQ結合型グルコース脱水素酵素がマルトースと反応すると 血糖 POCT装置は実際より高い血糖値を表示してしまう。このため、患者が不必要な インシュリン注射を行ってしまう結果、意識障害や昏睡状態に陥る等の低血糖事故が 発生し、大きな問題となっている。 [0005] However, the conventional PQQ-linked glucose dehydrogenase has the disadvantage that it is poor in stability and also reacts with manoleose and galactose. Maltose is a sugar used for infusion, and when PQQ-linked glucose dehydrogenase reacts with maltose. Blood glucose The POCT device displays blood glucose levels higher than actual. For this reason, as a result of unnecessary insulin injections by patients, hypoglycemia accidents such as disturbance of consciousness and falling into a coma have occurred, which is a major problem.
[0006] 特に現在の血糖 POCT装置の用途としては、単に簡易的に血糖を測る目的から、 さらに患者の自己管理及び治療の一手段としての重要性が高まっており、そのため に使用される自己血糖測定装置（Self-Monitoring of Blood Glucose:SMBG)の家庭 への普及は拡大の一途を迪つていることから、測定精度への要求性は非常に高いと 考えられる。  [0006] In particular, the present blood glucose POCT apparatus is used for the purpose of simply measuring blood sugar, and is becoming increasingly important as a means of patient self-management and treatment. Since the spread of measuring devices (Self-Monitoring of Blood Glucose: SMBG) to homes is steadily expanding, the demand for measurement accuracy is considered to be very high.
[0007] 現に、 2005年 2月には、 日本において、厚生労働省よりマルトース輸液ゃィコデキ ストリンを含む透析液を投与中の患者に対して、補酵素として PQQを利用している酵 素を用いた血糖測定器の使用に関し、注意を喚起する通達が出されている（2005 年 2月 7日；薬食安発第 0207005号など）。  [0007] Actually, in February 2005, an enzyme using PQQ as a coenzyme was used in Japan for a patient who was receiving a dialysate containing maltose infusion solution from the Ministry of Health, Labor and Welfare. A notice has been issued regarding the use of blood glucose meters (February 7, 2005; Drug Food Safety No. 0207005, etc.).
[0008] 一方、グルコースの脱水素反応を触媒し、 FADを補酵素とする補酵素結合型グノレ コース脱水素酵素としては、 Agrobacterium tumefaciens由来 (J. Biol. Chem. (1967) 2 42： 3665_3t '2)、 Cytophaga marinoflava由来 (Appl. Biochem. Biotechnol. (1996) 56 ： 301-310)、 Halomonas sp. a -15由来 (Enzyme Microb. Technol. (1998) 22： 269-27 4)、 Agaricus bisporus由来 (Arch. Microbiol.(1997) 167: 119-125, Appl. Microbiol. Bio technol. (1999)51 :58- 64)及び Macrol印 iota rhacodes由来 (Arch. Microbiol.(2001)176 : 178-186)の酵素が報告されている力 これらの酵素はグルコースの 2位及び/又は 3位の水酸基を酸化し、いずれもマルトースに対する作用性が高ぐグルコースに対 する選択性が低い。また、同じくマルトースへの作用性が高い、ブルクホルデリア 'セ パシァ（Burkholderia cepacia)由来の補酵素結合型グルコース脱水素酵素も知られ ているが、これは本来の天然型の酵素が α、 β、 γの 3種のサブユニットからなるへ テロオリゴマー酵素で、膜結合性酵素として知られている。したがって、酵素を得るに は可溶化の処理が必要であったり、クローユングで十分な活性を発現させる為には、 必要なサブユニットを同時にクローニングしなければならない等の課題があった。  [0008] On the other hand, as a coenzyme-linked type of glenolecide dehydrogenase that catalyzes the dehydrogenation reaction of glucose and uses FAD as a coenzyme, it is derived from Agrobacterium tumefaciens (J. Biol. Chem. (1967) 2 42: 3665_3t ' 2), derived from Cytophaga marinoflava (Appl. Biochem. Biotechnol. (1996) 56: 301-310), derived from Halomonas sp. A-15 (Enzyme Microb. Technol. (1998) 22: 269-27 4), derived from Agaricus bisporus (Arch. Microbiol. (1997) 167: 119-125, Appl. Microbiol. Bio technol. (1999) 51: 58-64) and Macrol-marked iota rhacodes (Arch. Microbiol. (2001) 176: 178-186) These enzymes have been reported to oxidize the 2nd and / or 3rd hydroxyl groups of glucose, both of which are less selective for glucose, which is more active on maltose. In addition, coenzyme-linked glucose dehydrogenase derived from Burkholderia cepacia, which is also highly effective against maltose, is also known. , A hetero-oligomer enzyme consisting of three subunits of γ, known as a membrane-bound enzyme. Therefore, solubilization treatment is necessary to obtain an enzyme, and necessary subunits must be cloned at the same time in order to express sufficient activity by cloning.
[0009] これに対して、本発明者らは、 FADを補酵素とする、膜結合型ではなレ、新規な可 溶性の補酵素結合型グルコース脱水素酵素をァスペルギルス.テレウスから精製して いる（特許文献 1)。この特許文献 1の補酵素結合型グルコース脱水素酵素は、ダル コースの 1位の水酸基を酸化し、グルコースに対する基質認識性に優れ、溶存酸素 の影響を受けず、し力もマルトースに対する作用性が低い（対グノレコース活性を 100 Q/oとした場合の対マルトース活性は 5。/0以下、対ガラクトース活性も 5%以下）というこ れまでに無い優れた特性を有するものである。 [0009] In contrast, the present inventors have purified a novel soluble coenzyme-linked glucose dehydrogenase using FAD as a coenzyme from Aspergillus terreus. (Patent Document 1). This coenzyme-linked glucose dehydrogenase of Patent Document 1 oxidizes the hydroxyl group at the 1-position of dalcose, has excellent substrate recognition for glucose, is not affected by dissolved oxygen, and has low strength against maltose. (vs. Gunorekosu activity versus maltose activity as a 100 Q / o is 5. / 0 or less, versus galactose activity than 5%) are those having excellent properties not in until now referred.
[0010] し力、しながら、この特許文献 1の補酵素結合型グルコース脱水素酵素は、野生の微 生物（例えばァスペルギルス属微生物など）の液体培養物から単離、抽出されたもの であり、その生産量には限りがあった。また、酵素生産量が極微量である上に、糖が 多量に酵素に結合し、通常の酵素に結合している N型や〇型糖鎖とは異なる種類の 糖に覆われた「糖包埋型酵素」とでも言うべき形態となっていることにより、その活性を 検出しにくいこと (酵素活性が低い）、糖鎖を酵素的あるいは化学的に除去できない こと、その結果、電気泳動において、通常のタンパク質染色（Coomassie Brilliant Blu e G-250等による）によりほとんど染色されず、遺伝子取得に必要な情報である酵素 のァミノ末端や内部のアミノ酸配列を解読することも通常の精製酵素からでは難しぐ 酵素遺伝子のクローニングに成功し、本酵素活性の発現を確認した事例は公知では ない。 However, the coenzyme-linked glucose dehydrogenase of Patent Document 1 is isolated and extracted from a liquid culture of a wild microorganism (for example, Aspergillus microorganism), The production was limited. In addition, the amount of enzyme produced is extremely small, and a large amount of sugar is bound to the enzyme, which is covered with a different type of sugar than the N-type or O-type sugar chains that are bound to normal enzymes. Because it is in a form that should be called "buried enzyme", its activity is difficult to detect (low enzyme activity), sugar chains cannot be removed enzymatically or chemically, and as a result, in electrophoresis, It is hardly stained by normal protein staining (by Coomassie Brilliant Blue G-250, etc.), and it is difficult to decode the amino terminal sequence of the enzyme and the internal amino acid sequence, which is the information necessary for gene acquisition, from ordinary purified enzymes. The case where the enzyme gene was successfully cloned and the expression of this enzyme activity was confirmed is not known.
[0011] 一方、ァスペルギルス'ォリゼ由来の補酵素結合型グルコース脱水素酵素について は 1967年にその存在が示唆されたことがあったが（非特許文献 1)、部分的に酵素 学的性質が明らかにされたのみで、マルトースに作用しないという特性は示唆されて いたにも関わらず、それ以降はァスペルギルス 'ォリゼ由来の補酵素結合型ダルコ一 ス脱水素酵素に関する詳細な報告はもちろんその他微生物由来の補酵素結合型グ ルコース脱水素酵素についても、グルコースの 1位の水酸基を酸化する酵素につい ては続報が無ぐ補酵素結合型グルコース脱水素酵素のアミノ酸配列や遺伝子に関 する報告も全く知られていなかった。  [0011] On the other hand, coenzyme-linked glucose dehydrogenase derived from Aspergillus oryzae was suggested to exist in 1967 (Non-patent Document 1), but the enzymological properties were partially clarified. Although it has been suggested that it does not act on maltose, a detailed report on coenzyme-bound dalcose dehydrogenase derived from Aspergillus olisee is of course available from other microorganisms. Regarding coenzyme-linked glucose dehydrogenase, there are no reports on the amino acid sequence or gene of coenzyme-linked glucose dehydrogenase, for which there is no further report on the enzyme that oxidizes the hydroxyl group at the 1-position of glucose. It wasn't.
[0012] また、グルコースデヒドロゲナーゼ EC 1. 1. 99. 10をグルコース測定に用いるアイ デァは知られていたが（特許文献 2参照）、 FAD結合型グルコース脱水素酵素が実 用的なレベルで生産されたことはなぐ実際にセンサに利用され実用化されるには至 つていなかった。その理由は、本酵素の菌体内での活性は微弱であり、菌体外に分 泌されてもその量は極僅かで、し力も多量の糖に覆われており活性が弱ぐ検出する のさえ困難であったために、遺伝子をクローニングできなかったためと推察される。 [0012] Further, although an idea using glucose dehydrogenase EC 1. 1. 99. 10 for glucose measurement has been known (see Patent Document 2), FAD-bound glucose dehydrogenase is at a practical level. What has been produced has not yet been put to practical use as a sensor. The reason for this is that the activity of this enzyme in the cells is weak and is not distributed outside the cells. Even if it is produced, the amount was very small, and the force was covered with a large amount of sugar, and it was difficult to detect the weak activity. Therefore, it was assumed that the gene could not be cloned.
[0013] 特許文献 1：国際公開第 2004/058958号パンフレット  [0013] Patent Document 1: Pamphlet of International Publication No. 2004/058958
特許文献 2：特開昭 59-25700号公報  Patent Document 2: JP 59-25700 A
非特許文献 1： Biochem.Biophys.Acta.，139,277-293,1967  Non-patent document 1: Biochem. Biophys. Acta., 139,277-293, 1967
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0014] PQQ結合型グルコース脱水素酵素の改変に関する遺伝子工学的方法に関しては 既に多くの技術が知られており、これらの従来技術は、主に該酵素の基質特異性の 低さや安定性の低さといつた従来の PQQ結合型グノレコース脱水素酵素の欠点を改 良するための改変型 PQQ結合型グノレコース脱水素酵素と、それを遺伝子工学的に 作成するための改変型遺伝子材料を提供している。  [0014] Many genetic engineering methods relating to modification of PQQ-linked glucose dehydrogenase are already known, and these conventional techniques mainly have low substrate specificity and low stability of the enzyme. We provide modified PQQ-linked gnolecose dehydrogenase to improve the shortcomings of conventional PQQ-linked gnolecose dehydrogenase and modified genetic material to create it genetically. .
[0015] し力、しながら、改変型遺伝子材料を用いて作成した改変型の PQQ結合型グルコース 脱水素酵素の場合には、依然としてグルコースに対する作用性を 100%とした際の マルトースへの作用性が概ね 10。 /。より高かったり、あるいはマルトースへの反応性を 低くさせた結果として、本来のグノレコースへの反応性 (比活性)までもが落ちてしまい 、基質十分量の条件で電気化学的な測定法で活性を見るとグルコースセンサとして の機能は不充分で、 POCT装置等への使用には至っていないのが実情である。更 に、 PQQ結合型グルコース脱水素酵素の活性発現に必要な補酵素 PQQは、広く一 般的に組換え宿主として用いられる大腸菌では作られず、 PQQを生産する宿主微 生物（シユードモナス等）に限定して組換え体を作らなければならないという問題もあ つに。  [0015] However, in the case of the modified PQQ-linked glucose dehydrogenase prepared using the modified genetic material, the activity on maltose when the activity on glucose is still 100% Is roughly 10. /. As a result of higher or lower reactivity to maltose, the original reactivity to gnorecose (specific activity) is also reduced, and the activity is measured by electrochemical measurement under the condition of sufficient substrate. Looking at it, the function as a glucose sensor is inadequate, and it is actually not used for POCT devices. In addition, coenzyme PQQ, which is necessary for the expression of PQQ-linked glucose dehydrogenase activity, is not made in E. coli, which is generally used as a recombinant host, and is limited to host microorganisms (such as Pseudomonas) that produce PQQ. As a result, there is a problem that a recombinant must be made.
[0016] 従って、本発明は、上記課題を解決し、グルコースに対する反応性、熱安定性、基 質認識性に優れ、し力もマルトースに対する作用性が低いという優れた特性を有する FAD結合型グルコース脱水素酵素をコードする新規な遺伝子（ポリヌクレオチド）、該 遺伝子により組換えられた形質転換細胞を用いる該酵素の製造方法、並びに、得ら れた該酵素を使用することを特徴とするグノレコースの測定方法、グルコース測定試薬 組成物、及びグノレコース測定用のバイオセンサ等を提供することを目的とする。 課題を解決するための手段 [0016] Therefore, the present invention solves the above-mentioned problems, and has excellent properties such as excellent reactivity to glucose, heat stability, and substrate recognition property, and low force and low activity on maltose. Novel gene (polynucleotide) encoding elementary enzyme, method for producing the enzyme using transformed cells recombined with the gene, and measurement of gno-lecose using the obtained enzyme The object is to provide a method, a glucose measuring reagent composition, a biosensor for measuring gnolecose, and the like. Means for solving the problem
[0017] 本発明者は、上記課題を解決すべく鋭意研究の結果、ァスペルギルス *ォリゼ (Aspe rgillus oryzae)菌株において、 FAD結合型グノレコース脱水素酵素が有意に発現する には、その遺伝子がコードするポリペプチドに、アミノ酸配列 (AGVPWV)を含んでい ることが必要であることを見出し、更に、その内の少なくとも 1個のアミノ酸が欠失した 場合には活性が実質的に失われることを確認し、本発明を完成した。即ち、本発明 は以下の各態様に係るものである。  [0017] As a result of diligent research to solve the above-mentioned problems, the present inventor encodes the gene to express FAD-bound gnolecose dehydrogenase significantly in Aspergillus oryzae strains. We have found that it is necessary to include the amino acid sequence (AGVPWV) in the polypeptide, and that the activity is substantially lost if at least one amino acid is deleted. The present invention has been completed. That is, the present invention relates to the following aspects.
[0018] [態様 1]アミノ酸配列： X1-X2-X3-X4-X5-X6  [0018] [Aspect 1] Amino acid sequence: X1-X2-X3-X4-X5-X6
(XI及び X2は脂肪族アミノ酸、 X3及び X6は分岐アミノ酸、並びに、 X4及び X5は複 素環式アミノ酸又は芳香族アミノ酸を示す）を含むポリペプチドから成る FAD結合型 グノレコース脱水素酵素をコードするポリヌクレオチド。  (XI and X2 are aliphatic amino acids, X3 and X6 are branched amino acids, and X4 and X5 are polycyclic amino acids or aromatic amino acids). Polynucleotide.
[態様 2]以下の（a)、 (b)又は（c)のポリペプチドをコードするポリヌクレオチド：  [Aspect 2] A polynucleotide encoding a polypeptide of the following (a), (b) or (c):
(a)配列番号 1に示されるアミノ酸配列から成るポリペプチド、  (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b)アミノ酸配列（a)のアミノ酸配列において、 1個〜数個のアミノ酸が置換、欠失又 は付加されたアミノ酸配列から成り、 FAD結合型グルコース脱水素酵素活性を有する ポリペプチド、又は  (b) a polypeptide having an FAD-linked glucose dehydrogenase activity consisting of an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence of amino acid sequence (a), or
(c)アミノ酸配列（a)と 70%以上の相同性を有するアミノ酸配列力 成り、かつ、 FAD 結合型グルコース脱水素酵素活性を有するポリペプチド。  (c) A polypeptide comprising an amino acid sequence having 70% or more homology with the amino acid sequence (a) and having FAD-bound glucose dehydrogenase activity.
[態様 3]以下の（d)、 (e)又は (f )のポリヌクレオチド：  [Aspect 3] The following polynucleotide (d), (e) or (f):
(d)配列番号 2又は配列番号 3に示される塩基配列を含むポリヌクレオチド、  (d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3,
(e)塩基配列（d)から成るポリヌクレオチドと相補的な塩基配列からなるポリヌクレオ チドとストリンジヱントな条件下でハイブリダィズし、かつ、 FAD結合型グルコース脱水 素酵素活性を有するポリペプチドをコードするポリヌクレオチド、又は  (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence (d) and encodes a polypeptide having FAD-linked glucose dehydrase activity Or
(f)塩基配列（d)から成るポリヌクレオチドと 70%以上の相同性を有する塩基配列を 含み、かつ、 FAD結合型グルコース脱水素酵素活性を有するポリペプチドをコードす るポリヌクレオチド。  (f) A polynucleotide encoding a polypeptide comprising a nucleotide sequence having 70% or more homology with the polynucleotide comprising the nucleotide sequence (d) and having FAD-linked glucose dehydrogenase activity.
[態様 4]アミノ酸配列: AGVPWVをコードする塩基配列力 成るセンスプライマー及 びァスペルギルス.ォリゼ（Aspergillus oryzae)由来の FAD結合型グルコース脱水素 酵素をコードするポリヌクレオチドの 3'末端側の塩基配列から成るリバースプライマ 一、又は、アミノ酸配列: AGVPWVをコードする塩基配列に対するアンチセンスプライ マー及びァスペルギルス ·ォリゼ（Aspergillus oryzae;由 5kのト AD結合型グノレコ^ ~ス 脱水素酵素をコードするポリヌクレオチドの 5'末端側の塩基配列から成るフォワード プライマーの組み合わせを用いる PCRによって増幅可能な DNA断片を有する、 FA D結合型グルコース脱水素酵素活性を有するポリペプチドをコードするポリヌクレオチ ド。 [Aspect 4] Amino acid sequence: Sense primer comprising nucleotide sequence that encodes AGVPWV and FAD-bound glucose dehydrogenation derived from Aspergillus oryzae Reverse primer consisting of the nucleotide sequence at the 3 'end of the polynucleotide encoding the enzyme, or an amino acid sequence: antisense primer against the nucleotide sequence encoding AGVPWV and Aspergillus oryzae (5k AD binding) A DNA fragment that can be amplified by PCR using a combination of forward primers consisting of a 5'-end base sequence of a polynucleotide encoding a type de noreco ^ ~ dehydrogenase. It has FAD-linked glucose dehydrogenase activity. A polynucleotide encoding a polypeptide.
[態様 5]アミノ酸配列: AGVPWVをコードする塩基配列力 成るプローブとストリンジ ェントな条件下でハイブリダィズし、かつ、 FAD結合型グルコース脱水素酵素活性を 有するポリペプチドをコードするポリヌクレオチド。  [Aspect 5] Amino acid sequence: A polynucleotide that hybridizes under stringent conditions with a probe having a nucleotide sequence that encodes AGVPWV and encodes a polypeptide having FAD-linked glucose dehydrogenase activity.
[態様 6] D_グルコースに対する酵素活性値を 100%とした場合、マルトースに対す る酵素活性値が 10 %以下、 D—ガラクトースに対する酵素活性値が 5 %以下である ことを特徴とする、ァスペルギルス'オリゼ（Aspergillus oryzae)由来の、 FAD結合型グ ルコース脱水素酵素をコードするポリヌクレオチド。  [Aspect 6] Aspergillus, wherein the enzyme activity value for D_glucose is 100%, the enzyme activity value for maltose is 10% or less, and the enzyme activity value for D-galactose is 5% or less. 'Polynucleotide encoding FAD-linked glucose dehydrogenase derived from Aspergillus oryzae.
[態様 7] 300U/mg以上の酵素活性を有することを特徴とする、ァスペルギルス.ォ リゼ（Aspergillus oryzae)由来の FAD結合型グルコース脱水素酵素をコードするポリ ヌクレオチド。  [Aspect 7] A polynucleotide encoding a FAD-linked glucose dehydrogenase derived from Aspergillus oryzae, characterized by having an enzyme activity of 300 U / mg or more.
[態様 8]上記のポリヌクレオチドを保有する組換えベクター。  [Aspect 8] A recombinant vector having the polynucleotide described above.
[態様 9]上記の組換えベクターを用いることによって作成された形質転換細胞。  [Aspect 9] A transformed cell produced by using the above recombinant vector.
[態様 10]上記の形質転換細胞を培養し、得られた培養物から、グルコースを脱水素 する作用を有する FAD結合型グルコース脱水素酵素を採取することを特徴とする FA [Aspect 10] FA characterized in that FAD-conjugated glucose dehydrogenase having an action of dehydrogenating glucose is collected from the culture obtained by culturing the above transformed cells.
D結合型グルコース脱水素酵素の製造方法。 A method for producing D-linked glucose dehydrogenase.
[態様 11]上記の記載のポリヌクレオチドにコードされる、組換え FAD結合型ダルコ一 ス脱水素酵素。  [Aspect 11] A recombinant FAD-binding dalcos dehydrogenase encoded by the polynucleotide described above.
[態様 12]上記の FAD結合型グルコース脱水素酵素を使用することを特徴とするダル コースの測定方法。  [Aspect 12] A method for measuring dalcose, wherein the FAD-bound glucose dehydrogenase is used.
[態様 13]上記の FAD結合型グルコース脱水素酵素を含有することを特徴とするダル コース測定試薬組成物。 [態様 14]上記の FAD結合型グルコース脱水素酵素を使用することを特徴とするダル コース測定用のバイオセンサ。 [Aspect 13] A reagent composition for measuring dalcose, which contains the above-mentioned FAD-linked glucose dehydrogenase. [Aspect 14] A biosensor for measuring dalcose, characterized by using the FAD-bound glucose dehydrogenase described above.
発明の効果  The invention's effect
[0019] 本発明のポリヌクレオチドを利用することにより、グルコースに対する基質認識性に 優れ、し力、もマルトースに対する作用性が低いとレ、う優れた特性を有する FAD結合型 グノレコース脱水素酵素を、例えば、遺伝子組み換え技術により均質かつ大量に生産 すること力 S可言 となる。  [0019] By using the polynucleotide of the present invention, a FAD-bound gnolecose dehydrogenase having excellent substrate recognizability to glucose, excellent strength, and low activity against maltose is obtained. For example, the ability to produce homogeneous and large-scale products using genetic recombination technology is S.
また、このように生産された酵素は、 FAD結合型グルコース脱水素酵素で問題とな つていた糖の量を目的に応じてコントロールできるため、糖含量を減らした酵素を調 製することで、血糖測定などにおいて、試料中の糖 (グルコースなど）に対する作用 性を変えることも可能である。  In addition, the enzyme produced in this way can control the amount of sugar, which has been a problem with FAD-bound glucose dehydrogenase, according to the purpose, so by preparing an enzyme with reduced sugar content, In blood glucose measurement, etc., it is possible to change the action on sugars (such as glucose) in the sample.
図面の簡単な説明  Brief Description of Drawings
[0020] [図 1]酵素固定化電極によるグルコース濃度の検量線を示す。  [0020] FIG. 1 shows a calibration curve of glucose concentration with an enzyme-immobilized electrode.
[図 2]PCRによる目的遺伝子の検出結果を示す。図中の記号は以下の通り。 M : 200b p DNAラダーマーカー（タカラバイオ社製） 1：ァスペルギルス'オリゼ NBRC42682：ァ スペルギルス .ォリゼ NBRC53753：ァスペルギルス 'ォリゼ NBRC62154：ァスペルギル ス'ォリゼ NBRC41815：ァスペルギルス *ォリゼ NBRC42206：ァスペルギルス *ォリゼ N BRC100959  [Figure 2] Shows the detection result of the target gene by PCR. The symbols in the figure are as follows. M: 200 bp DNA ladder marker (manufactured by Takara Bio Inc.) 1: Aspergillus oryzae NBRC42682: Aspergillus oryzae NBRC53753: Aspergillus oryzae NBRC62154: Aspergillus oryzae NBRC41815: Aspergillus * oryzae NBRC42206: Aspergillus NBCRC
[図 3]サザンハイブリダィゼーシヨンによる目的遺伝子の検出結果を示す。図中の記 号は図 2と同じである。  [Fig. 3] Shows the detection result of the target gene by Southern hybridization. The symbols in the figure are the same as in Figure 2.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0021] 本発明の FAD結合型グノレコース脱水素酵素においてはアミノ酸配列： Xト X2-X3-X 4-X5-X6 [0021] In the FAD-linked gnolecose dehydrogenase of the present invention, the amino acid sequence is: X, X2-X3-X 4-X5-X6
(XI及び X2は同一又は互いに異なる脂肪族アミノ酸、 X3及び X6は同一又は互い に異なる分岐アミノ酸、並びに、 X4及び X5は同一又は互いに異なる複素環式ァミノ 酸又は芳香族アミノ酸を示す）、即ち、 6個のアミノ酸力 成るポリペプチドが含まれて レ、ることが技術的に重要な点の一つであり、その為に、該酵素が菌体内で有意に発 現される。尚、発現した該酵素は必ずしも菌体外に分泌される必要はなぐ菌体内に 留まる場合もある。対照的に、本明細書の実施例に具体的に示されるように、アミノ酸 配列全体の相同性等から FAD結合型グルコース脱水素酵素と考えられる酵素をコー ドする遺伝子であっても、該アミノ酸配列から成るポリペプチドをコードしてレ、なレ、遺 伝子は FAD結合型グルコース脱水素酵素活性を有する蛋白質を発現しなレ、。 (XI and X2 are the same or different aliphatic amino acids, X3 and X6 are the same or different branched amino acids, and X4 and X5 are the same or different heterocyclic amino acids or aromatic amino acids), It is one of the technically important points that a polypeptide consisting of 6 amino acids is contained, and for this reason, the enzyme is significantly expressed in the microbial cells. The expressed enzyme does not necessarily need to be secreted outside the cell. It may stay. In contrast, as specifically shown in the examples of the present specification, even a gene encoding an enzyme considered to be FAD-linked glucose dehydrogenase from the homology of the entire amino acid sequence, etc. The gene encodes a polypeptide consisting of a sequence, and the gene does not express a protein having FAD-linked glucose dehydrogenase activity.
[0022] 上記 6個のアミノ酸配列は、好ましくは、 FAD結合型グルコース脱水素酵素であるポ リペプチドの 202〜207番目に位置し、又は、 X1〜X6の少なくとも一つ力 XIがァラ ニン (A)、 X2がグリシン（G)、 X3がバリン（V)、 X4がプロリン（P)、 X5がトリプトファン (W )、又は、 X6がバリン (V)である。例えば、その好適例として、アミノ酸配列： AGVPWV (配列番号 4)を挙げること力できる。  [0022] The six amino acid sequences are preferably located at positions 202 to 207 of a polypeptide that is a FAD-linked glucose dehydrogenase, or at least one force XI of X1 to X6 is alanine ( A), X2 is glycine (G), X3 is valine (V), X4 is proline (P), X5 is tryptophan (W), or X6 is valine (V). For example, the amino acid sequence AGVPWV (SEQ ID NO: 4) can be cited as a preferred example.
[0023] 本発明において、「FAD結合型グルコース脱水素酵素」とは、電子受容体存在下で 、グルコースの 1位の水酸基を脱水素（酸化）する反応を触媒し、グルコースへの作 用性に対してマルトースへの作用性が 10%以下である可溶性の蛋白質を意味し、該 酵素は以下の性質を特徴とする。  [0023] In the present invention, "FAD-linked glucose dehydrogenase" refers to catalyzing a reaction of dehydrogenating (oxidizing) the hydroxyl group at the 1-position of glucose in the presence of an electron acceptor, and thereby acting on glucose. In contrast, it means a soluble protein having an action on maltose of 10% or less, and the enzyme is characterized by the following properties.
1)フラビンアデニンジヌクレオチド (FAD)を補酵素とする、  1) Flavin adenine dinucleotide (FAD) as a coenzyme,
2)酸素を電子受容体としない、及び  2) oxygen is not an electron acceptor, and
3)グルコースへの作用性に対してマルトースへの作用性が 10%以下である。  3) Maltose activity is 10% or less compared to glucose activity.
[0024] 本発明の FAD結合型グルコース脱水素酵素の中で、アミノ酸配列： AGVPWVを有 するものとしては、特に、ァスペルギルス'オリゼ（Aspergillus oryzae)由来のものが好 ましレ、。その代表的な菌株として以下の表 1に示されるような、 NBRC5375株、 NBR C4079株、 NBRC4203株、 NBRC4214株、 NBRC4268株、 NBRC5238株、 N BRC6215株、 NBRC30104株 及び、 NBRC30113株等を挙げることが出来る。 アミノ酸配列： AGVPWVは、該酵素のアミノ酸配列において、シグナル配列部分の開 始アミノ酸 Mを 1番目とした時の第 202〜207番目（NBRC5375株由来）付近（その 他の菌株由来の酵素の場合には、その位置に相当する場所）に含まれている。 [0024] Among the FAD-linked glucose dehydrogenases of the present invention, those having the amino acid sequence AGVPWV are particularly preferably those derived from Aspergillus oryzae. Examples of such strains include NBRC5375, NBR C4079, NBRC4203, NBRC4214, NBRC4268, NBRC5238, NBRC6215, NBRC30104, and NBRC30113 as shown in Table 1 below. I can do it. Amino acid sequence: AGVPWV is the vicinity of 202-207 (from NBRC5375 strain) when the starting amino acid M of the signal sequence portion is the first in the amino acid sequence of the enzyme (in the case of enzymes derived from other strains). Is included in the location corresponding to the position).
[0025] 例えば、ァスペルギルス'オリゼ（Aspergillus oryzae) NBRC5375株が発現する FAD 結合型グノレコース脱水素酵素のアミノ酸配列は配列番号 1 (シグナルペプチドを含む[0025] For example, the amino acid sequence of the FAD-linked gnolecose dehydrogenase expressed by Aspergillus oryzae NBRC5375 is SEQ ID NO: 1 (including signal peptide)
)、それをコードする染色体 DNAの塩基配列は配列番号 2、又、配列番号 1に示され るアミノ酸に対応する cDNAは配列番号 3で夫々示される。尚、配列番号 2又は 3に おいて、アミノ酸配列： AGVPWVをコードする塩基配列は GCTGGTGTTCCATGGGT T (配列番号 5)である。 ), The nucleotide sequence of the chromosomal DNA encoding it is SEQ ID NO: 2, and the cDNA corresponding to the amino acid shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, respectively. In SEQ ID NO: 2 or 3, The base sequence encoding the amino acid sequence AGVPWV is GCTGGTGTTCCATGGGT T (SEQ ID NO: 5).
[0026] 従って、本発明のポリヌクレオチドは、ァスペルギルス'ォリゼ（Aspergillus oryzae)の 菌株由来の上記のものに加えて、以下の（a)、 （b)又は（c)のポリペプチドをコードす るポリヌクレ才チド：  [0026] Therefore, the polynucleotide of the present invention encodes the following polypeptide (a), (b) or (c) in addition to the above-mentioned one derived from the strain of Aspergillus oryzae. Polynucle tide:
(a)配列番号 1に示されるアミノ酸配列から成るポリペプチド、  (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b)アミノ酸配列（a)において、 1個〜数個のアミノ酸が置換、欠失又は付加されたァ ミノ酸配列から成り、 FAD結合型グルコース脱水素酵素活性を有するポリペプチド、 又は  (b) a polypeptide comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in amino acid sequence (a) and having FAD-linked glucose dehydrogenase activity, or
(c)アミノ酸配列（a)と 70%以上の相同性を有するアミノ酸配列力 成り、かつ、 FAD 結合型グルコース脱水素酵素活性を有するポリペプチド、が含まれる。  (c) a polypeptide having an amino acid sequence having 70% or more homology with the amino acid sequence (a) and having FAD-linked glucose dehydrogenase activity.
[0027] 更に、本発明のポリヌクレオチドには、以下の（d)、（e)又は（f)のポリヌクレオチド：  [0027] Furthermore, the polynucleotide of the present invention includes the following polynucleotide (d), (e) or (f):
(d)配列番号 2又は配列番号 3に示される塩基配列を含むポリヌクレオチド、  (d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3,
(e)塩基配列（d)から成るポリヌクレオチドと相補的な塩基配列からなるポリヌクレオド とストリンジェントな条件下でハイブリダィズし、かつ、 FAD結合型グルコース脱水素酵 素活性を有するポリペプチドをコードするポリヌクレオチド、又は  (e) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence (d) under stringent conditions and encodes a polypeptide having FAD-linked glucose dehydrogenase activity. Nucleotides, or
(f)塩基配列（d)から成るポリヌクレオチドと 70%以上の相同性を有する塩基配列を 含み、かつ、 FAD結合型グルコース脱水素酵素活性を有するポリペプチドをコードす るポリヌクレオチド、が含まれる。  (f) a polynucleotide encoding a polypeptide having a nucleotide sequence having 70% or more homology with a polynucleotide comprising the nucleotide sequence (d) and having FAD-linked glucose dehydrogenase activity .
[0028] 特に、上記の（b)又は（c)のポリペプチドが上記アミノ酸配列： X1-X2-X3-X4-X5-X6 を含むもの、又は、（e)又は（f)のポリヌクレオチドが該アミノ酸配列をコードする塩基 配列を含むものが好ましレ、。更に、このアミノ酸配列が AGVPWVであるものが好まし レ、。  [0028] In particular, the polypeptide of (b) or (c) above comprises the amino acid sequence: X1-X2-X3-X4-X5-X6, or the polynucleotide of (e) or (f) Those containing a base sequence encoding the amino acid sequence are preferred. Furthermore, it is preferable that this amino acid sequence is AGVPWV.
[0029] 本明細書において、 70%以上の相同性を有するアミノ酸配列又は塩基配列とは、夫 々比較対象となる基準配列の全長にわたり、少なくとも 70%の同一性を示し、好まし くは 75%以上、より好ましくは 80%以上、さらにより好ましくは 90%以上、特に好まし くは 95%以上の同一性を有する各配列をいう。このような配列の同一性パーセンテ ージは、基準配列を照会配列として比較するアルゴリズムをもった公開又は市販され ているソフトウェアを用いて計算することができる。例として、 BLAST、 FASTA、又 は GENETYX (ソフトウェア開発株式会社製）などを用いることができ、これらはデフ オルトパラメーターで使用することができる。 [0029] In the present specification, an amino acid sequence or a base sequence having 70% or more homology shows at least 70% identity over the entire length of the reference sequence to be compared, and preferably 75%. % Or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more. Such sequence identity percentages are published or commercially available with algorithms that compare a reference sequence as a query sequence. Can be calculated using software. For example, BLAST, FASTA, or GENETYX (manufactured by Software Development Co., Ltd.) can be used, and these can be used with default parameters.
[0030] 本発明において、ポリヌクレオチド間のハイブリダィズに際しての「ストリンジヱントな 条件下でハイブリダィズ」の具体的な条件とは、例えば、 50。/。ホノレムアミド、 5 X SSC (150mM 塩ィ匕ナトリウム、 15mM クェン酸三ナトリウム、 10mM リン酸ナトリウム 、 ImM エチレンジァミン四酢酸、 ρΗ7. 2)、 5 Χデンハート（Denhardt' s)溶液、 0. 1 % SDS、 10% デキストラン硫酸及び 100 z g/mLの変性サケ精子 DNAで 42 °Cインキュベーションした後、フィルターを 0. 2 X SSC中 42°Cで洗浄することを例示 すること力 Sできる。 In the present invention, specific conditions for “hybridization under stringent conditions” for hybridizing between polynucleotides are, for example, 50. /. Honolemamide, 5 X SSC (150 mM sodium chloride, 15 mM trisodium citrate, 10 mM sodium phosphate, ImM ethylenediamine tetraacetic acid, ρΗ7.2), 5Χ Denhardt's solution, 0.1% SDS, 10 Exemplify washing the filter at 42 ° C in 0.2 X SSC after 42 ° C incubation with% dextran sulfate and 100 zg / mL denatured salmon sperm DNA.
[0031] 更に、本発明のポリヌクレオチドは、アミノ酸配列： AGVPWVをコードする塩基配列か ら成るセンスプライマー及びァスペルギルス.ォリゼ（Aspergillus oryzae)由来の FAD 結合型グノレコース脱水素酵素をコードするポリヌクレオチドの 3 '末端側の塩基配列 力 成るリバースプライマー、又は、アミノ酸配列： AGVPWVをコードする塩基配列対 するアンチセンスプライマー及びァスペルギルス'オリゼ（Aspergillus oryzae)由来の FAD結合型グルコース脱水素酵素をコードするポリヌクレオチドの 5'末端側の塩基 配列力 成るフォワードプライマーの組み合わせを用いる PCRによって増幅可能な D NA断片を有する、 FAD結合型グルコース脱水素酵素活性を有するポリペプチドをコ ードするポリヌクレオチドを含む。  [0031] Further, the polynucleotide of the present invention includes a sense primer comprising a base sequence encoding amino acid sequence: AGVPWV, and a polynucleotide encoding a FAD-bound gnolecose dehydrogenase derived from Aspergillus oryzae. A reverse primer consisting of a nucleotide sequence on the terminal side, or an antisense primer for the amino acid sequence: AGVPWV-encoding nucleotide sequence and a polynucleotide encoding a FAD-linked glucose dehydrogenase derived from Aspergillus oryzae A polynucleotide encoding a polypeptide having FAD-linked glucose dehydrogenase activity, which has a DNA fragment that can be amplified by PCR using a combination of forward primers consisting of a base primer having a 5'-end base sequence.
[0032] 或いは、本発明のポリヌクレオチドは、アミノ酸配列： AGVPWVをコードする塩基配列 から成るプローブとストリンジヱントな条件下でハイブリダィズし、かつ、 FAD結合型グ ルコース脱水素酵素活性を有するポリペプチドをコードするポリヌクレオチドを含む。  [0032] Alternatively, the polynucleotide of the present invention hybridizes with a probe comprising the nucleotide sequence encoding the amino acid sequence: AGVPWV under stringent conditions and encodes a polypeptide having FAD-bound glucose dehydrogenase activity. Polynucleotides.
[0033] 好ましくは、アミノ酸配列： AGVPWVをコードする塩基配列は（GCTGGTGTTCCATG GGTT)である。又、上記の PCR及びストリンジヱントな条件下でのハイブリダィズに関 する各種の条件は本明細書中の実施例の記載に準じて当業者が適宜選択すること が出来る。  [0033] Preferably, the base sequence encoding the amino acid sequence AGVPWV is (GCTGGTGTTCCATG GGTT). Further, various conditions relating to the above-mentioned PCR and hybridization under stringent conditions can be appropriately selected by those skilled in the art according to the description of the examples in the present specification.
[0034] 更に、本発明のポリヌクレオチドには、 D—グノレコースに対する酵素活性値を 100% とした場合、マルトースに対する酵素活性値が 10%以下、好ましくは 5%以下、より好 ましくは 3%以下であり、 D—ガラクトースに対する酵素活性値が 5%以下、好ましくは[0034] Furthermore, the polynucleotide of the present invention has an enzyme activity value for maltose of 10% or less, preferably 5% or less, more preferably, when the enzyme activity value for D-gnolecose is 100%. The enzyme activity value for D-galactose is 5% or less, preferably 3% or less, preferably
3%以下、より好ましくは 2%以下、更に好ましくは 1 %以下である、 FAD結合型 グノレコース脱水素酵素をコードするポリヌクレオチド、又はタンパク質当たりの比活性 力 ¾OOU/mg以上、好ましくは 500UZmg以上、より好ましくは 1， OOOUZmg以上 の酵素活性を有する FAD結合型グルコース脱水素酵素をコードするポリヌクレオチド が含まれる。尚、ここで、「タンパク質当たりの比活性」とは、例えば、本明細書の実施 例 7に記載のような、培養上清を濃縮し SDS— PAGEで単一バンドとして確認された 状態で測定されたものである。 3% or less, more preferably 2% or less, and even more preferably 1% or less, a polynucleotide encoding FAD-linked gnolecose dehydrogenase, or a specific activity per protein ¾OOU / mg or more, preferably 500 UZmg or more, More preferably, a polynucleotide encoding an FAD-linked glucose dehydrogenase having an enzyme activity of 1, OOOUZmg or more is included. Here, “specific activity per protein” is measured, for example, in a state where the culture supernatant is concentrated and confirmed as a single band by SDS-PAGE, as described in Example 7 of the present specification. It has been done.
[0035] 尚、本発明において、「ポリヌクレオチド」とは、プリン又はピリミジンが糖に /3 -N-ダリ コシド結合したヌクレオシドのリン酸エステル (ATP (アデノシン三リン酸）、 GTP (グァ ノシン三リン酸）、 CTP (シチジン三リン酸）、 UTP (ゥリジン三リン酸）；又は dATP (デ ォキシアデノシン三リン酸）、 dGTP (デォキシグアノシン三リン酸）、 dCTP (デォキシ シチジン三リン酸）、 dTTP (デォキシチミジン三リン酸））が 100個以上結合した分子 を言い、具体的には FAD結合型グノレコース脱水素酵素をコードする染色体 DNA、 染色体 DNAから転写された mRNA、 mRNAから合成された cDNA及び、それらを 铸型として PCR増幅したポリヌクレオチドを含む。「オリゴヌクレオチド」とはヌクレオチ ドが 2-99個連結した分子を言う。また「ポリペプチド」とは、アミド結合 (ペプチド結合） 又は非天然の残基連結によって互いに結合した 30個以上のアミノ酸残基から構成さ れた分子を意味し、さらには、これらに糖鎖が付加したものや、人工的に化学的修飾 がなされたもの等も含む。  [0035] In the present invention, "polynucleotide" means a nucleoside phosphate ester (ATP (adenosine triphosphate), GTP (guanosine triphosphate) in which purine or pyrimidine is bonded to a sugar by / 3-N-daricoside. Phosphate), CTP (cytidine triphosphate), UTP (uridine triphosphate); or dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate) ), DTTP (deoxythymidine triphosphate)) is a molecule that binds more than 100 molecules. Specifically, it is synthesized from chromosomal DNA that encodes FAD-linked gnolecose dehydrogenase, mRNA transcribed from chromosomal DNA, and mRNA. It includes cDNA and polynucleotides that have been PCR-amplified using them as saddles. “Oligonucleotide” refers to a molecule in which 2-99 nucleotides are linked. “Polypeptide” means a molecule composed of 30 or more amino acid residues linked to each other by amide bonds (peptide bonds) or unnatural residue linkages. Includes those that have been added or those that have been artificially chemically modified.
[0036] 本発明のポリヌクレオチド (遺伝子）の最も具体的な態様は、配列番号 2又は配列 番号 3の塩基配列を含むポリヌクレオチドである。配列番号 2に代表される染色体 D NAであるポリヌクレオチドは、例えば、ァスペルギルス 'ォリゼ NBRC5375株力、ら染 色体 DNAライブラリーを調製し、特許文献 1に記載のァスペルギルス'テレウス由来 の FAD結合型グルコース脱水素酵素の N末端及び内部配列のアミノ酸をエドマン法 等によって決定して得られるアミノ酸配列、及び、 spergillus oryzaeのゲノム解析 プロジェクト」の成果として、 2006年 1月に DOGAN (Database of the Genomes Analy zed at NITE) (ウェブサイト http://www. bio.nite.go.jp/dogan/Top)で公開されたァス ペルギルス.ォリゼ（NBRC100959株）のゲノム配列情報に基づいて作成した複数 のオリゴヌクレオチドプローブを用いて当業者に公知の方法によって上記染色体 DN Aライブラリーをスクリーニングすることによって取得することができる。 [0036] The most specific embodiment of the polynucleotide (gene) of the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3. A polynucleotide that is a chromosomal DNA represented by SEQ ID NO: 2 is prepared, for example, aspergillus oryzae NBRC5375 strain, a chromosomal DNA library, and the FAD-binding type derived from Aspergillus terreus described in Patent Document 1. In January 2006, DOGAN (Database of the Genomes Analyze) was developed as a result of the `` Amino acid sequence obtained by determining the N-terminal and internal amino acids of glucose dehydrogenase by the Edman method and the genome analysis project of spergillus oryzae ''. zed at NITE) (Website http://www.bio.nite.go.jp/dogan/Top) It can be obtained by screening the above chromosomal DNA library by a method known to those skilled in the art using a plurality of oligonucleotide probes prepared on the basis of the genome sequence information of Pergillus oryzae (NBRC100959 strain).
[0037] プローブの標識は、当業者に公知の任意の方法、例えば、ラジオアイソトープ (RI) 法又は非 RI法によって行うことができる力 非 RI法を用いることが好ましい。非 RI法と しては、蛍光標識法、ピオチン標識法、化学発光法等が挙げられるが、蛍光標識法 を用いることが好ましい。蛍光物質としては、オリゴヌクレオチドの塩基部分と結合で きるものを適宜に選択して用いることができる力 シァニン色素（例えば、 Cy DyeT Mシリーズの Cy3、 Cy5等）、ローダミン 6G試薬、 N-ァセトキシ -N2 -ァセチルァミノ フルオレン (AAF)、 AAIF (AAFのヨウ素誘導体）などを使用することができる。  [0037] The labeling of the probe is preferably performed by any method known to those skilled in the art, for example, a force non-RI method that can be performed by a radioisotope (RI) method or a non-RI method. Examples of the non-RI method include a fluorescence labeling method, a piotine labeling method, a chemiluminescence method, and the like, but it is preferable to use a fluorescence labeling method. Fluorescent substances can be selected from those that can bind to the base moiety of the oligonucleotide as appropriate. Cyanine dyes (eg, Cy DyeTM M series Cy3, Cy5, etc.), rhodamine 6G reagent, N-acetoxy- N2 -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), etc. can be used.
[0038] 或いは、配列番号 3に代表される cDNAであるポリヌクレオチドは、例えば、本明細 書の実施例に具体的に記載されているように、 cDNAライブラリーを铸型とし、上記 で作成したオリゴヌクレオチドプライマー（プローブ）のセットを用いた当業者に公知の 各種 PCR法によって、もしくはァスペルギルス 'ォリゼ NBRC5375株から抽出した全 RNAもしくは mRNAを铸型とする RT-PCR法によっても得ることができる。尚、プラ イマ一を設計する場合には、プライマーのサイズ (塩基数）は铸型 DNAとの間の特異 的なアニーリングを満足させることを考慮し、 15-40塩基、望ましくは 15-30塩基であ る。ただし、 LA (long and accurate) PCRを行う場合には、少なくとも 30塩基が効 率的である。センス鎖（5 '末端側）とアンチセンス鎖（3'末端側）からなる一組あるい は一対（2本）のプライマーが互いにァニールしないよう、両プライマー間の相補的配 歹 IJを避けるようにする。さらに、铸型 DNAとの安定な結合を確保するため GC含量を 約 50%にし、プライマー内において GC- richあるいは AT-richが偏在しないように する。アニーリング温度は Tm (melting temperature)に依存するので、特異性の高い PCR産物を得るため、 Tm値が 55-65°Cで互いに近似したプライマーを選定する。ま た、 PCRにおけるプライマー使用の最終濃度が約 0. 1から約 Ι μ Μになるよう調整 する等を留意することも必要である。また、プライマー設計用の市販のソフトウェア、 例えば OligoTM [National Bioscience Inc. (米国）製]、 GENETYX (ソフトウェア 開発株式会社製)等を用レ、ることもできる。 [0039] 尚、このようなオリゴヌクレオチドプローブやオリゴヌクレオチドプライマーセットは、例 えば本発明のポリヌクレオチドである cDNAを適当な制限酵素で切断して作成するこ とちできる。 [0038] Alternatively, the polynucleotide represented by SEQ ID NO: 3 was prepared as described above using a cDNA library as a saddle shape, as specifically described in the examples of the present specification, for example. It can also be obtained by various PCR methods known to those skilled in the art using a set of oligonucleotide primers (probes), or by RT-PCR using total RNA or mRNA extracted from Aspergillus oryzae NBRC5375 strain as a saddle type. When designing a primer, the size of the primer (number of bases) should be 15-40 bases, preferably 15-30 bases, considering that it will satisfy the specific annealing with the vertical DNA. It is. However, for LA (long and accurate) PCR, at least 30 bases are efficient. Avoid complementary alignment IJ between both primers so that the primer pair of sense strand (5 'end side) and antisense strand (3' end side) or pair (two) primer does not anneal to each other. To. In addition, the GC content should be about 50% to ensure stable binding to the truncated DNA, and GC-rich or AT-rich should not be unevenly distributed in the primer. Since the annealing temperature depends on Tm (melting temperature), in order to obtain highly specific PCR products, select primers that are close to each other with a Tm value of 55-65 ° C. It is also necessary to pay attention to the final concentration of primers used in PCR so that the final concentration is about 0.1 to about 約 μΙ. Also, commercially available software for primer design such as Oligo ™ [National Bioscience Inc. (USA)], GENETYX (Software Development Co., Ltd.), etc. can be used. [0039] It should be noted that such an oligonucleotide probe or oligonucleotide primer set can be prepared, for example, by cleaving cDNA, which is a polynucleotide of the present invention, with an appropriate restriction enzyme.
[0040] 又、本発明のポリヌクレオチドは、例えば、前記のァスペルギルス 'ォリゼ NBRC53 75株由来の FAD結合型グルコース脱水素酵素 cDNAを、公知のミューテーシヨン導 入法や変異導入 PCR法等によって改変して作成することができる。更に、 NBRC53 75株以外のァスペルギルス'オリゼ菌株の染色体 DNAやその cDNAライブラリーか ら、配列番号 1のヌクレオチド配列情報に基づいて作成したオリゴヌクレオチドを用い るプローブハイブリダィゼーシヨン法によって取得することができる。ハイブリダィゼー シヨンに際して、ストリンジヱント条件を様々に変化させることによって、上記ポリヌクレ ォチドを取得することができる。ストリンジヱント条件は、ハイブリダィゼーシヨン及び洗 浄工程における塩濃度、有機溶媒 (ホルムアルデヒド等）の濃度、温度条件等によつ て規定され、例えば、米国特許 No.6, 100,037号明細書等に開示されているような、当 業者らに周知の様々な条件を採用することができる。  [0040] The polynucleotide of the present invention is modified, for example, from the above-mentioned FAD-linked glucose dehydrogenase cDNA derived from Aspergillus oryzae NBRC5375 strain by a known mutation introduction method or mutagenesis PCR method. Can be created. Furthermore, it must be obtained from the chromosomal DNA of Aspergillus oryzae strains other than the NBRC5375 strain or its cDNA library by the probe hybridization method using the oligonucleotide prepared based on the nucleotide sequence information of SEQ ID NO: 1. Can do. In hybridization, the polynucleotide can be obtained by variously changing the stringent conditions. Stringent conditions are defined by the concentration of salt in the hybridization and washing process, the concentration of organic solvent (formaldehyde, etc.), temperature conditions, etc., for example, as described in US Pat. No. 6,100,037, etc. Various conditions known to those skilled in the art, such as those disclosed, can be employed.
[0041] 更に、文献（例えば Carruthers(1982)Cold Spring Harbor Symp. Quant. Biol. 47:411- 418;Adams(1983)J. Am. Chem. Soc. 105:661; Belousov(1997)Nucleic Acid Res. 25:3 440-3444; Frenkel(1995)Free Radic. Biol. Med. 19:373- 380;Blommers(1994)Bioche mistry 33:7886-7896; Narang(1979)Meth. Enzymol. 68:90;Brown(1979)Meth. Enzym ol. 68: 109; Beaucage(1981)Tetra. Lett. 22: 1859; 米国特許第 4,458, 066号）に記載 されているような周知の化学合成技術により、 in vitroにおいて本発明のポリヌクレオ チドを合成することができる。  [0041] Further, literature (for example, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47: 411-418; Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acid Res 25: 3 440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Bloomers (1994) Bioche mistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; US Pat. No. 4,458, 066). The polynucleotides of the invention can be synthesized.
[0042] 本発明の組換えベクターは、クローニングベクター又は発現ベクターであり、インサ ートとしてのポリヌクレオチドの種類や、その使用目的等に応じて適宜のものを使用 する。例えば、 cDNA又はその ORF領域をインサートとして FAD結合型グルコース脱 水素酵素を生産する場合には、 in vitro転写用の発現ベクターや、大腸菌、枯草菌 等の原核細胞、酵母、カビなどの糸状菌、昆虫細胞、哺乳動物細胞等の真核細胞の それぞれに適した発現ベクターを使用することもできる。  [0042] The recombinant vector of the present invention is a cloning vector or an expression vector, and an appropriate one is used according to the type of polynucleotide as an insert, the purpose of use, and the like. For example, when producing FAD-linked glucose dehydrogenase using cDNA or its ORF region as an insert, expression vectors for in vitro transcription, prokaryotic cells such as Escherichia coli and Bacillus subtilis, filamentous fungi such as yeast and mold, Expression vectors suitable for eukaryotic cells such as insect cells and mammalian cells can also be used.
[0043] 本発明の形質転換細胞としては、例えば、大腸菌、枯草菌等の原核細胞や、酵母 、カビ、昆虫細胞、哺乳動物細胞等の真核細胞等を使用することができる。これらの 形質転換細胞は、電気穿孔法、リン酸カルシウム法、リボソーム法、 DEAEデキストラ ン法など公知の方法によって組換えベクターを細胞に導入することによって調製する ことができる。組換えベクター及び形質転換細胞の具体例として、下記実施例に示し た組換えベクターと、このベクターによる形質転換大腸菌及び形質転換カビが挙げら れる。 [0043] Examples of the transformed cell of the present invention include prokaryotic cells such as Escherichia coli and Bacillus subtilis, and yeast. , Eukaryotic cells such as molds, insect cells, mammalian cells and the like can be used. These transformed cells can be prepared by introducing a recombinant vector into cells by a known method such as electroporation, calcium phosphate method, ribosome method, DEAE dextran method. Specific examples of the recombinant vector and the transformed cell include the recombinant vector shown in the Examples below, and transformed Escherichia coli and transformed mold using the vector.
[0044] 本発明の FAD結合型グノレコース脱水素酵素を大腸菌などの微生物で DNAを発現 させて生産させる場合には、微生物中で複製可能なオリジン、プロモーター、リボソ ーム結合部位、 DNAクローニング部位、ターミネータ一配列等を有する発現べクタ 一に前記のポリヌクレオチドを組換えた発現ベクターを作成し、この発現ベクターで 宿主細胞を形質転換したのち、得られた形質転換体を培養すれば、 FAD結合型グ ルコース脱水素酵素を微生物で大量生産することができる。この際、任意の翻訳領 域の前後に開始コドンと停止コドンを付加して発現させれば、任意の領域を含む FA D結合型グルコース脱水素酵素断片を得ることもできる。あるいは、他の蛋白質との 融合蛋白質として発現させることもできる。この融合蛋白質を適当なプロテアーゼで 切断することによつても目的とする FAD結合型グルコース脱水素酵素を取得すること ができる。大腸菌用発現ベクターとしては、 pUC系、 pBluescriptll, pET発現シス テム、 pGEX発現システム、 pCold発現システムなどが例示できる。  [0044] When the FAD-linked gnolecose dehydrogenase of the present invention is produced by expressing DNA in a microorganism such as Escherichia coli, the origin, promoter, ribosome binding site, DNA cloning site capable of replicating in the microorganism, If an expression vector is prepared by recombining the above-mentioned polynucleotide into an expression vector having a terminator sequence, etc., host cells are transformed with this expression vector, and the resulting transformant is cultured. Type glucose dehydrogenase can be mass-produced with microorganisms. At this time, if a start codon and a stop codon are added before and after an arbitrary translation region and expressed, an FAD-linked glucose dehydrogenase fragment containing the arbitrary region can be obtained. Alternatively, it can be expressed as a fusion protein with another protein. The target FAD-bound glucose dehydrogenase can also be obtained by cleaving this fusion protein with an appropriate protease. Examples of the expression vector for E. coli include pUC system, pBluescriptll, pET expression system, pGEX expression system, and pCold expression system.
[0045] 或いは、 FAD結合型グルコース脱水素酵素を真核細胞で発現させて生産させる場 合には、前記ポリヌクレオチドを、プロモーター、スプライシング領域、ポリ（A)付加部 位等を有する真核細胞用発現ベクターに挿入して組換えベクターを作成し、真核細 胞内に導入すれば、 FAD結合型グルコース脱水素酵素を真核細胞で生産すること ができる。プラスミドのような状態で細胞内に維持することもできるし、染色体中に組 みこませて維持することもできる。発現ベクターとしては、 pKAl、 pCDM8、 pSVK3 、 pSVL、 pBK-CMV、 pBK— RSV、 EBVベクター、 pRS、 pYE82などが例示できる 。また、 pIND/V5- His、 pFLAG- CMV_2、 pEGFP_Nl、 pEGFP-Clなどを発 現ベクターとして用いれば、 Hisタグ、 FLAGタグ、 GFPなど各種タグを付加した融合 蛋白質として FAD結合型グルコース脱水素酵素ポリペプチドを発現させることもでき る。真核細胞としては、サル腎臓細胞 COS_7、チャイニーズハムスター卵巣細胞 CH Oなどの哺乳動物培養細胞、出芽酵母、分裂酵母、カビ、カイコ細胞、アフリカッメガ エル卵細胞などが一般に用いられる力 FAD結合型グルコース脱水素酵素を発現で きるものであれば、レ、かなる真核細胞でもよい。発現ベクターを真核細胞に導入する には、電気穿孔法、リン酸カルシウム法、リボソーム法、 DEAEデキストラン法など公 知の方法を用いることができる。 [0045] Alternatively, when FAD-linked glucose dehydrogenase is expressed in eukaryotic cells for production, the polynucleotide is a eukaryotic cell having a promoter, a splicing region, a poly (A) addition site, etc. FAD-linked glucose dehydrogenase can be produced in eukaryotic cells by creating a recombinant vector by inserting it into an expression vector for use and introducing it into a eukaryotic cell. It can be maintained in a cell like a plasmid, or it can be maintained in a chromosome. Examples of expression vectors include pKAl, pCDM8, pSVK3, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYE82. In addition, if pIND / V5-His, pFLAG-CMV_2, pEGFP_Nl, pEGFP-Cl, etc. are used as expression vectors, FAD-linked glucose dehydrogenase poly- hydrase can be used as a fusion protein to which various tags such as His tag, FLAG tag, and GFP are added. Peptides can also be expressed The Eukaryotic cells are commonly used for cultured mammalian cells such as monkey kidney cell COS_7, Chinese hamster ovary cell CH 2 O, budding yeast, fission yeast, mold, silkworm cell, Xenopus egg cell, etc. FAD-coupled glucose dehydrogenation Any eukaryotic cell may be used as long as it can express the enzyme. To introduce the expression vector into eukaryotic cells, known methods such as electroporation, calcium phosphate method, ribosome method, DEAE dextran method can be used.
[0046] 特に、ァスペルギルス.ォリゼ（Aspergillus oryzae)由来の本発明の FAD結合型グル コース脱水素酵素をコードするポリヌクレオチドを保有する組換えベクターで、適当な ァスペルギルス'オリゼ株を形質転換するセルフクローユングが好適である。  [0046] In particular, a recombinant vector carrying a polynucleotide encoding the FAD-linked glucose dehydrogenase of the present invention derived from Aspergillus oryzae, and a self-clone for transforming an appropriate Aspergillus oryzae strain. Jung is preferred.
[0047] FAD結合型グルコース脱水素酵素を原核細胞や真核細胞で発現させた後、培養 物（菌体、もしくは菌体外に分泌された酵素を含む培養液、培地組成物等）から目的 蛋白質を単離精製するためには、公知の分離操作を組み合わせて行うことができる。 例えば、尿素などの変性剤や界面活性剤による処理、熱処理、 pH処理、超音波処 理、酵素消化、塩析ゃ溶媒沈殿法、透析、遠心分離、限外濾過、ゲル濾過、 SDS-P AGE,等電点電気泳動、イオン交換クロマトグラフィー、疎水性クロマトグラフィー、 逆相クロマトグラフィー、ァフィ二ティークロマトグラフィー（タグ配列を利用した方法及 び FAD補酵素結合型グルコース脱水素酵素に特異的なポリクローナル抗体、モノク ローナル抗体を用いる方法も含む）、などが挙げられる。  [0047] After expressing FAD-conjugated glucose dehydrogenase in prokaryotic cells or eukaryotic cells, the target is obtained from a culture (a culture solution or a medium composition containing an enzyme secreted outside the cell). In order to isolate and purify the protein, known separation operations can be combined. For example, treatment with denaturing agents and surfactants such as urea, heat treatment, pH treatment, ultrasonic treatment, enzyme digestion, salting out solvent precipitation method, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-P AGE , Isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, affinity chromatography (tag sequence-based methods and polyclonal antibodies specific to FAD coenzyme-linked glucose dehydrogenase) Antibody, and a method using a monoclonal antibody).
[0048] また、 FAD結合型グルコース脱水素酵素は、本発明のポリヌクレオチド（cDNA又 はその翻訳領域）を用いた組換え DNA技術によって取得できる。例えば前記ポリヌ クレオチドを有するベクターから in vitro転写によって RNAを調製し、これを铸型と して in vitro翻訳を行うことにより in vitroで FAD結合型グルコース脱水素酵素を作 成すること力 Sできる。またポリヌクレオチドを公知の方法により適当な発現ベクターに 組換えれば、大腸菌、枯草菌等の原核細胞や、酵母、カビ、昆虫細胞、哺乳動物細 胞等の真核細胞で、ポリヌクレオチドがコードしている FAD結合型グノレコース脱水素 酵素を大量に発現させる事ができる。また宿主に対応して、同一アミノ酸配列である 力 コドンユーセージを最適化したポリブクレオチドを導入しても良レ、。また糖鎖の要[0048] In addition, FAD-linked glucose dehydrogenase can be obtained by a recombinant DNA technique using the polynucleotide (cDNA or translation region thereof) of the present invention. For example, it is possible to prepare FAD-linked glucose dehydrogenase in vitro by preparing RNA from the above-mentioned polynucleotide-containing vector by in vitro transcription, and performing in vitro translation using this as a truncated form. If the polynucleotide is recombined into an appropriate expression vector by a known method, the polynucleotide is encoded in prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, mold, insect cells and mammalian cells. It is possible to express a large amount of the FAD-linked gnolecose dehydrogenase. It is also possible to introduce a polynucleotide optimized for force codon usage, which is the same amino acid sequence, according to the host. The key to sugar chains
、不要、その他のペプチド修飾の必要性に応じて、適宜宿主は選択することができる [0049] FAD結合型グルコース脱水素酵素を in vitro発現させて生産させる場合には、前記 のポリヌクレオチドを、 RNAポリメラーゼが結合できるプロモーターを有するベクター に揷入して組換えベクターを作成し、このベクターを、プロモーターに対応する RNA ポリメラーゼを含むゥサギ網状赤血球溶解物や小麦胚芽抽出物などの in vitro翻訳 系に添加すれば、 FAD結合型グルコース脱水素酵素を in vitroで生産することがで きる。 RNAポリメラーゼが結合できるプロモーターとしては、 T3、 T7、 SP6などが例 示できる。これらのプロモーターを含むベクターとしては、 pKAl、 pCDM8、 pT3/ Τ718、 ρΤ7/319、 pBluescriptllなどが例示できる。 Depending on the necessity of other peptide modifications, the host can be selected as appropriate. [0049] When producing FAD-linked glucose dehydrogenase by in vitro expression, a recombinant vector is prepared by inserting the polynucleotide described above into a vector having a promoter to which RNA polymerase can bind. FAD-linked glucose dehydrogenase can be produced in vitro by adding the vector to an in vitro translation system such as a rabbit reticulocyte lysate or wheat germ extract containing RNA polymerase corresponding to the promoter. Examples of promoters to which RNA polymerase can bind include T3, T7 and SP6. Examples of the vector containing these promoters include pKAl, pCDM8, pT3 / Τ718, ρΤ7 / 319, and pBluescriptll.
[0050] 本発明の組換え FAD結合型グノレコース脱水素酵素は以上に記載された方法で製 造すること力 Sできる。このような FAD結合型グノレコース脱水素酵素は、電子受容体存 在下でグノレコースを脱水素する反応を触媒する酵素であるから、この反応による変化 が利用できる用途であれば、特に制限されない。例えば、生体物質を含む試料中の グルコースの測定及び測定用試薬、消去用試薬へ使用するなどの医療分野、臨床 分野への使用が可能であり、補酵素結合型グノレコース脱水素酵素を使用した物質 生産においても使用可能である。  [0050] The recombinant FAD-linked gnolecose dehydrogenase of the present invention can be produced by the method described above. Such a FAD-bound type of gonorlecose dehydrogenase is an enzyme that catalyzes the reaction of dehydrogenating gnolecose in the presence of an electron acceptor, and is not particularly limited as long as the change due to this reaction can be used. For example, substances that can be used in the medical field and clinical field such as measuring and measuring reagents for glucose in samples containing biological materials, as well as reagents for erasing, and using coenzyme-linked gnorolecose dehydrogenase It can also be used in production.
[0051] 本発明のグルコース測定試薬組成物は、全てを混合して単一の試薬としてもよぐ 相互に干渉する成分が存在する場合には、各成分を適宜な組み合せとなる様に分 割してもよい。また、これらは、溶液状、もしくは粉末状試薬として調製してもよぐさら にこれらを濾紙もしくはフィルムなどの適当な支持体に含有させ試験紙もしくは分析 用フィルムとして調製してもよい。なお、過塩素酸などの除タンパク剤ゃグノレコース定 量を含有する標準試薬を添付してもよい。本組成物中の酵素の量は、 1試料当り 0. 1から 50単位程度が好ましい。グノレコースを定量するための検体は、例えば血漿、血 清、髄液、唾液、尿などが挙げられる。  [0051] The reagent composition for glucose measurement of the present invention may be mixed together to form a single reagent. When there are components that interfere with each other, the components are separated so that they are combined appropriately. May be. These may be prepared as a solution or powdery reagent, and may be prepared as a test paper or an analytical film by containing them in a suitable support such as filter paper or film. A standard reagent containing a deproteinizing agent such as perchloric acid or a quantitative amount of gnolecose may be attached. The amount of enzyme in the composition is preferably about 0.1 to 50 units per sample. Examples of the specimen for quantifying gnolecose include plasma, serum, spinal fluid, saliva, urine and the like.
[0052] 本発明のバイオセンサは、酵素として本発明の FAD結合型グルコース脱水素酵素 を含む反応層に使用し、試料液中のグルコース濃度を測定するグルコースセンサで ある。例えば、絶縁性基板上にスクリーン印刷などの方法を利用して作用極、その対 極及び参照極からなる電極系を形成し、この電極系上に接して親水性高分子と酸化 還元酵素と電子受容体とを含む酵素反応層を形成することによって作製される。この バイオセンサの酵素反応層上に基質を含む試料液を滴下すると、酵素反応層が溶 解して酵素と基質が反応し、これにともなって電子受容体が還元される。酵素反応終 了後、還元された電子受容体を電気化学的に酸化させ、このとき、このノィォセンサ は得られる酸化電流値から試料液中の基質濃度を測定することが可能である。また、 この他に、発色強度あるいは pH変化などを検知する方式のバイオセンサも構築可能 である。 [0052] The biosensor of the present invention is a glucose sensor that is used in a reaction layer containing the FAD-bound glucose dehydrogenase of the present invention as an enzyme and measures the glucose concentration in a sample solution. For example, an electrode system consisting of a working electrode, its counter electrode, and a reference electrode is formed on an insulating substrate using a method such as screen printing, and is in contact with the hydrophilic polymer and oxidized with this electrode system. It is produced by forming an enzyme reaction layer containing a reductase and an electron acceptor. When a sample solution containing a substrate is dropped onto the enzyme reaction layer of this biosensor, the enzyme reaction layer is dissolved and the enzyme reacts with the substrate, and the electron acceptor is reduced accordingly. After completion of the enzyme reaction, the reduced electron acceptor is electrochemically oxidized. At this time, the nanosensor can measure the substrate concentration in the sample solution from the obtained oxidation current value. In addition, it is possible to construct a biosensor that detects color intensity or pH change.
[0053] バイオセンサの電子受容体としては、電子の授受能に優れた化学物質を用いること ができる。電子の授受能に優れた化学物質とは、一般的に「電子伝達体」、「メデイエ ータ」あるいは「酸化還元媒介剤」と呼ばれる化学物質であり、これらに該当する化学 物質として、例えば、特表 2002-526759に挙げられた電子伝達体や酸化還元媒介 剤などを利用してもよい。具体的には、オスミウム化合物、キノン化合物、フヱリシアン 化合物等が挙げられる。  [0053] As an electron acceptor of a biosensor, a chemical substance excellent in electron transfer capability can be used. Chemical substances that excel in electron transfer are chemical substances that are generally called `` electron mediators '', `` mediators '', or `` redox mediators ''. The electron mediators and redox mediators listed in Special Table 2002-526759 may be used. Specific examples include an osmium compound, a quinone compound, and a fluoric compound.
[0054] FAD結合型グルコース脱水素酵素の活性測定においては、該酵素を、好ましくは終 濃度 0.：!〜 1. Ounit/mLになるように適宜希釈して用いる。なお、該酵素の酵素活 性単位 (unit)は 1分間に 1 μ molのグルコースを酸化する酵素活性である。本発明 の FAD結合型グノレコース脱水素酵素の酵素活性は、次の方法で測定できる。  [0054] In measuring the activity of FAD-linked glucose dehydrogenase, the enzyme is preferably diluted appropriately so that the final concentration is 0.:! To 1. Ounit / mL. The enzyme activity unit (unit) of the enzyme is an enzyme activity that oxidizes 1 μmol of glucose per minute. The enzyme activity of the FAD-linked gnolecose dehydrogenase of the present invention can be measured by the following method.
[0055] [酵素活性測定法]  [0055] [Enzyme activity measurement method]
0. 1M リン酸カリウム緩衝液（ρΗ7. 0) 1. OmL、 1. OM D—グノレコース 1. OmL 、 3mM 2, 6—ジクロロフェノールインドフエノール（以下 DCIPという） 0· 14mL、 3 mM 1—メトキシー 5—メチルフエナジゥムメチルサルフェイト 0. 2mL、水 0. 61mL を 3mL石英セル (光路長 lcm)に添カ卩し、恒温セルホルダー付き分光光度計にセッ トして 37°Cで 5分間インキュベート後、酵素溶液 0. 05mLを添加後、 DCIPの 600η mにおける吸光度変化（A ABSZmin)を測定する。 DCIPの pH7. 0におけるモル 吸光係数を 16. 3 X 103cm— 1とし、 1分間に 1 μ molの DCIPが還元される酵素活 性が実質的に該酵素活性 limitと等価であることから、吸光度変化より該酵素活性を 次式に従って求めた。 0.1 M potassium phosphate buffer (ρΗ7.0) 1. OmL, 1. OM D—Gnolecose 1. OmL, 3 mM 2,6-Dichlorophenol indophenol (DCIP) 0 · 14 mL, 3 mM 1—Methoxy 5—Methylphenmethyl methyl sulfate 0.2 mL, 0.61 mL of water is added to a 3 mL quartz cell (optical path length lcm), set in a spectrophotometer equipped with a thermostatic cell holder, and 5 ° C at 37 ° C. After incubation for 5 minutes, add 0.05 mL of the enzyme solution, and then measure the change in absorbance (A ABSZmin) at 600 ηm of DCIP. Since the molar extinction coefficient of DCIP at pH 7.0 is 16.3 X 10 3 cm— 1 , the enzyme activity that reduces 1 μmol of DCIP per minute is substantially equivalent to the enzyme activity limit. The enzyme activity was determined from the change in absorbance according to the following formula.
[0056] [数 1] mm it (mti^iiil)= x x 素の #«寧 [0056] [Equation 1] mm it (mti ^ iiil) = xx prime # «Ning
[0057] 本酵素のタンパク濃度の測定においては、該酵素を、好ましくは終濃度 0. 2〜0. 9 mg/mL になるように適宜希釈して用いる。本発明におけるタンパク濃度は、 日本 バイオ'ラッド (株）力ら購入できるタンパク濃度測定キットである Bio— Rad Protein Assayを用い、取扱説明書に従って、牛血清アルブミン (BSA，和光純薬工業 (株 )製，生化学用）を標準物質として作成した検量線力 換算して求めることができる。 [0057] In measuring the protein concentration of the present enzyme, the enzyme is preferably diluted as appropriate so that the final concentration is preferably 0.2 to 0.9 mg / mL. The protein concentration in the present invention was determined using the Bio-Rad Protein Assay, a protein concentration measurement kit that can be purchased from Japan Bio's Rad Co., Ltd., and according to the instruction manual, bovine serum albumin (BSA, Wako Pure Chemical Industries, Ltd.). It can be obtained by converting the calibration curve force created as a reference material.
[0058] 尚、本発明を実施するために使用する様々な技術は、特にその出典を明示した技 術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能 である。例えば、遺伝子工学及び分子生物学的技術は Sambrook and Maniatis, in M olecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, F. M. et al., Current Protocols in Molecular Biology, John Wil ey & Sons, New York, N.Y， 1995などに記載の方法あるいはそこで引用された文献 記載の方法又はそれらと実質的に同様な方法や改変法に基づき実施可能である。さ らに、この発明における用語は基本的には IUPAC-IUB Commission on Biochemical Nomenclatureによるものであり、あるいは当該分野において慣用的に使用される用語 の意味に基づくものである。  [0058] Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known documents, etc., except for the technique that clearly indicates the source. It is. For example, genetic engineering and molecular biology techniques include Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wil It can be carried out based on the method described in ey & Sons, New York, NY, 1995 or the like, the method described in the literature cited therein, or a method or modification method substantially similar thereto. Further, the terms in the present invention are basically based on the IUPAC-IUB Commission on Biochemical Nomenclature or based on the meanings of terms conventionally used in the field.
[0059] 以下、実施例に則して本発明を更に詳しく説明する。尚、本発明の技術的範囲は これらの記載によって何等制限されるものではなレ、。又、本明細書中に引用される文 献に記載された内容は、本明細書の一部として本明細書の開示内容を構成するもの である。  Hereinafter, the present invention will be described in more detail with reference to examples. It should be noted that the technical scope of the present invention is not limited in any way by these descriptions. In addition, the contents described in the documents cited in this specification constitute the disclosure content of this specification as a part of this specification.
実施例 1  Example 1
[0060] (ァスペルギルス.ォリゼ NBRC5375株由来 FAD結合型グルコース脱水素酵素と推定 される遺伝子の大腸菌へのクローニング）  [0060] (Cloning of a gene presumed to be FAD-linked glucose dehydrogenase from Aspergillus oryzae NBRC5375 strain into Escherichia coli)
(1)菌体培養  (1) Cell culture
グルコースけ力ライ社製） 1 % (W/V)、脱脂大豆（昭和産業社製） 2% (W/V)、 コーンスティープリカ一（サンエイ糖化社製） 0· 5% (W/V)、硫酸マグネシウム七水 和物（ナカライネ土製） 0. 1 % (W/V)及び水からなる液体培地を ρΗ6· 0に調整し、 1 OOmLを 500mL容の坂口フラスコに入れ、 121°C、 20分間オートクレーブした。冷 却したこの液体培地に、ァスペルギルス'ォリゼ（Aspergillus oryzae) NBRC5375株 を接種し、 28°Cで 48時間振とう培養した後、遠心分離機を用いて、湿菌体 15. 5gを 回収した。 1% (W / V), defatted soybean (made by Showa Sangyo) 2% (W / V), corn steep liquor (made by Sanei Saccharification Co., Ltd.) 0.5% (W / V) , Magnesium sulfate seven water A liquid medium composed of Japanese (Nakaraine soil) 0.1% (W / V) and water was adjusted to ρΗ6.0, and 1 OOmL was placed in a 500 mL Sakaguchi flask and autoclaved at 121 ° C for 20 minutes. The cooled liquid medium was inoculated with Aspergillus oryzae NBRC5375 strain and cultured with shaking at 28 ° C for 48 hours, and then 15.5 g of wet cells were collected using a centrifuge.
(2)ァスペルギルス 'ォリゼ NBRC 5375株の FAD結合型ダルコース脱水素酵素活 性確認  (2) Confirmation of FAD-bound dalcose dehydrogenase activity of Aspergillus oryzae NBRC 5375
(1)で得られた菌体を 50mMリン酸カリウム緩衝液 (PH7. 5)に懸濁し、海砂 B (ナ 力ライ社製）を用いて菌体を磨砕後、遠心して上清を回収し、無細胞抽出液とした。 前述の酵素活性測定法に従い、無細胞抽出液の FAD結合型グルコース脱水素酵 素活性を確認したところ、無細胞抽出液当たり、 0. 0043UZmLの FAD結合型グノレ コース脱水素酵素活性を確認した。 Suspend the cells obtained in (1) in 50 mM potassium phosphate buffer ( PH 7.5), grind the cells using sea sand B (Nagoryai Co., Ltd.), centrifuge and remove the supernatant. Was recovered and used as a cell-free extract. When the FAD-bound glucose dehydrogenase activity of the cell-free extract was confirmed according to the enzyme activity measurement method described above, 0.003 UZmL of FAD-bound glucose dehydrogenase activity was confirmed per cell-free extract.
(3)全 RNAの単離  (3) Total RNA isolation
(1)で得られた菌体のうち湿菌体 0. 31gを液体窒素により凍結した後、粉碎し、 IS OGEN (二ツボンジーン社製）を用いて全 RN Aを抽出した。  Of the bacterial cells obtained in (1), 0.31 g of wet cells were frozen with liquid nitrogen, then powdered, and total RNA was extracted using IS OGEN (Nitsubon Gene).
(4) RT— PCR  (4) RT—PCR
TaKaRa RNA LA PCR Kit(AMV)Ver.l. l (タカラバイオ社製）を使用し、下記条件で RT— PCRを行レ、、約 1. 8kbpの FAD結合型グルコース脱水素酵素と想定される遺 伝子を含む PCR産物を取得した。  Using TaKaRa RNA LA PCR Kit (AMV) Ver.l. (Takara Bio Inc.), RT-PCR was performed under the following conditions, and assumed to be about 1.8 kbp FAD-conjugated glucose dehydrogenase. A PCR product containing the gene was obtained.
テンプレート ：（3)で抽出した全 RNA Template: Total RNA extracted in (3)
プライマー ： Primer :
プライマー 1 ： 5 ' -tgggatcctatgctcttctcactggcat-3 ' (酉己歹 lj番号 6)  Primer 1: 5 '-tgggatcctatgctcttctcactggcat-3' (酉 己 歹 lj number 6)
フフイマ1 ~ 2 ： 5 -gccaagcttctaagcactcttcgcatcctccttaatcaagtc-3 己歹 亏, J 尚、プライマー 1及び 2は、上記の DOGAN (Database of the Genomes Analyzed at N ITE) (ウェブサイト http：〃 www.bio.nite.go.jp/dogan/Top)で公開されているァスペル ギノレス-ォ];ゼ NBRC100959株の遺伝子角军析結果より A〇090005000449 (「コリ ン脱水素酵素」と推定されている）の塩基配列を元に合成した。 Fufuima 1 ~ 2: 5 -gccaagcttctaagcactcttcgcatcctccttaatcaagtc-3 himself歹亏, J It should be noted, primers 1 and 2, the above DOGAN (Database of the Genomes Analyzed at N ITE) ( web site http: 〃 www.bio.nite.go. jp / dogan / Top) published by Asper Ginoles-;]; NBRC100959 strain based on the results of gene angle analysis, based on the base sequence of A090005000449 (presumed to be “Colin dehydrogenase”) Synthesized.
その理由は、本発明者等により見出されたァスペルギルス *テレウスの FAD結合型グ ルコース脱水素酵素遺伝子の塩基配列情報に基づき、上記 AO090005000449力 S コリン脱水素酵素遺伝子ではなく、ァスペルギルス 'ォリゼの FAD結合型グルコース 脱水素酵素遺伝子と推測された為である。 The reason for this is that the Aspergillus * Teleus FAD-coupled group found by the present inventors. This is because, based on the nucleotide sequence information of the Lucose dehydrogenase gene, it was assumed that it was not the AO090005000449 S choline dehydrogenase gene but the Aspergillus oryzae FAD-linked glucose dehydrogenase gene.
反応条件 ：逆転写反応 42°C、 30分（1サイクル） Reaction conditions: Reverse transcription reaction 42 ° C, 30 minutes (1 cycle)
変性 99°C、 5分 （1サイクル） Denaturation 99 ° C, 5 minutes (1 cycle)
冷却 5°C、 5分（1サイクル） Cooling 5 ° C, 5 minutes (1 cycle)
変性 94°C、 2分（1サイクル） Denaturation 94 ° C, 2 minutes (1 cycle)
変性 94。C、 30秒、アニーリング 45°C、 30秒、伸長反応 72。C、 1分 30秒（25サイクノレ ) Denaturation 94. C, 30 seconds, annealing 45 ° C, 30 seconds, extension reaction 72. C, 1 minute 30 seconds (25 cyclores)
伸長反応 72°C、 5分（1サイクル） Extension reaction 72 ° C, 5 minutes (1 cycle)
(5) FAD結合型グルコース脱水素酵素と推定される遺伝子を含むプラスミドの調製 (5) Preparation of plasmid containing gene presumed to be FAD-linked glucose dehydrogenase
(4)で得られた PCR増幅断片を制限酵素 BamHIと Hindlllで切断し、同制限酵素 処理した PUC18ベクター（タカラバイオ社製）に、 DNA Ligation Kit Ver.2.1 (タカラバ ィォ社製）を用いてライゲーシヨンし、 FAD結合型グルコース脱水素酵素と推定される 遺伝子を含むプラスミドを調製した。 The PCR amplification fragment obtained in (4) was cleaved with restriction enzymes BamHI and Hindlll, and DNA Ligation Kit Ver.2.1 (Takara Bio) was used for the PUC18 vector (Takara Bio) treated with the same restriction enzymes. And a plasmid containing a gene presumed to be FAD-linked glucose dehydrogenase was prepared.
(6)形質転換体の作製  (6) Production of transformants
(5)で得られたプラスミドを Ε· coli JM109 Competent Cell (タカラバイオ社製 )に導入して形質転換した。アンピシリンナトリウム（和光純薬社製)含有の LBプレー トで、 37°Cでー晚培養した後、ダイレクト PCRにて、生育したコロニー 1個に、 FAD結 合型グノレコース脱水素酵素と推定される遺伝子を含んだプラスミドが導入されている ことを確認し、アンピシリンナトリウム含有の LBプレートで形質転換体を取得した。 実施例 2  The plasmid obtained in (5) was introduced into E. coli JM109 Competent Cell (Takara Bio Inc.) for transformation. LB plate containing ampicillin sodium (manufactured by Wako Pure Chemical Industries, Ltd.), cultured at 37 ° C, and then directly PCR is estimated to be FAD-conjugated gnorecose dehydrogenase After confirming that the plasmid containing the gene had been introduced, transformants were obtained using ampicillin sodium-containing LB plates. Example 2
(ァスペルギルス.ォリゼ NBRC5375株由来 FAD結合型グルコース脱水素酵素と推定 される遺伝子のァスペルギルス'ォリゼへのクローニング）  (Cloning of a gene presumed to be FAD-linked glucose dehydrogenase from Aspergillus oryzae NBRC5375 strain into Aspergillus oryzae)
(1)染色体 DNAの抽出  (1) Chromosomal DNA extraction
実施例 1の（1)で得られた湿菌体のうち 0. 25gを液体窒素により凍結した後、粉碎 し、常法により染色体 DNAを抽出した。  Of the wet cells obtained in Example 1 (1), 0.25 g was frozen with liquid nitrogen, ground, and chromosomal DNA was extracted by a conventional method.
(2) FAD結合型グルコース脱水素酵素と推定される遺伝子のクローニング 使用する宿主としては、ァスペルギルス 'ォリゼ NS4株を使用した。本菌株は、公知 文献 l (Biosci. Biotech. Biochem.,61(8)，1367_1369, 1997)にあるように、 1997年（平成 9年）に醸造試験所で育種され、転写因子の解析、各種酵素の高生産株の育種など に利用され、分譲されているものが入手可能である。 (2) Cloning of a gene presumed to be FAD-linked glucose dehydrogenase As the host to be used, Aspergillus oryzae NS4 strain was used. This strain was bred in a brewery laboratory in 1997 (Heisei 9) as described in the well-known literature l (Biosci. Biotech. Biochem., 61 (8), 1367_1369, 1997), and analyzed transcription factors, Those that are used for breeding high-producing strains of enzymes and are distributed are available.
本菌株に対し、公知文献 2 (Aspergillus属の異種遺伝子発現系、峰時俊貴、化学と 生物、 38、 12、 P831- 838、 2000)に記載してあるァスペルギルス.ォリゼ由来のァミラ ーゼ系の改良プロモーターを使用し、その下流に（1)で得られた染色体 DNAを錡 型として、 DOGAN (Database of the Genomes Analyzed at NITE) (ウェブサイト http:〃而 w.bio.nite.go.jp/dogan/Top)で公開されている AO090005000449の塩 基配列を元に合成した以下のプライマー：  For this strain, the amylase system derived from Aspergillus oryzae described in known literature 2 (Aspergillus genus heterologous gene expression system, Toshiki Mineki, Chemistry and Biology, 38, 12, P831-838, 2000). DOGAN (Database of the Genomes Analyzed at NITE) (website http: metaphysical w.bio.nite.go.jp/ The following primers were synthesized based on the base sequence of AO090005000449 published in dogan / Top):
1. genelr :  1.genelr:
5 -(acecgtcgac tgaccaattccgcagctcgtcaaaatgctcttctcactggcattcctga-3 (酉己歹 I [番号 8) 5-(acecgtcgac tgaccaattccgcagctcgtcaaaatgctcttctcactggcattcctga-3 (酉 己 歹 I [number 8)
2. genelR: 2.genelR:
5— ggctgaactaattcctcctacgcttctcacgaat gtg)— 3 (酉己歹1 号 9) 5— ggctgaactaattcctcctacgcttctcacgaat gtg) — 3 (Tsubaki 1 No. 9)
(Fは 5 '側、 Rは 3 '側、括弧内：制限酵素切断部位、下線部： enoA 5 ' _UTR、その他: ORF)  (F is 5 'side, R is 3' side, in parentheses: restriction enzyme cleavage site, underlined: enoA 5 '_UTR, others: ORF)
を用いて増幅した FAD結合型グノレコース脱水素酵素と推定される遺伝子を結合させ ることで、本遺伝子が発現可能なベクターを調製した。 A vector capable of expressing this gene was prepared by binding a gene presumed to be a FAD-linked gnolecose dehydrogenase amplified using the above.
形質転換は、基本的には公知文献 2及び公知文献 3 (清酒用麹菌の遺伝子操作技 術、五味勝也、醸協、 P494-502, 2000)に記載の方法に準じて実施することで形質転 換体を取得した。  Transformation is basically carried out according to the method described in known literature 2 and publicly known literature 3 (gene manipulation technology for koji mold for sake, Katsuya Gomi, Shukyo, P494-502, 2000). Acquired.
比較例 Comparative example
(ァスペルギルス.ォリゼ NBRC100959株由来 FAD結合型グルコース脱水素酵素と推 定される遺伝子（AO090005000449)のァスペルギルス'ォリゼへのクローニング） (1)菌体培養  (Cloning of FAD-linked glucose dehydrogenase gene (AO090005000449) from Aspergillus oryzae NBRC100959 strain into Aspergillus oryzae) (1) Cell culture
グノレコース 1。/0 (W/V)、脱脂大豆 2% (W/V)、コーンスティープリカ一 0· 5% ( W/V)、硫酸マグネシウム七水和物 0. 1 % (W/V)及び水からなる液体培地を pH 6. 0に調整し、 lOOmLを 500mL容の坂口フラスコに入れ、 121°C、 20分間オートク レーブした。冷却したこの液体培地に、ァスペルギルス'オリゼ NBRC100959株を 接種し、 28°Cで 48時間振とう培養した後、遠心分離機を用いて、菌体 10. 5gを回収 した。 Gnore Course 1. / 0 (W / V), defatted soybeans 2% (W / V), corn steep liquor 0.5% (W / V), magnesium sulfate heptahydrate 0.1% (W / V) and water Adjust the liquid medium to pH 6.0, place lOOmL in a 500mL Sakaguchi flask, autoclaved at 121 ° C for 20 minutes. It was rave. The cooled liquid medium was inoculated with Aspergillus oryzae NBRC100959 strain and cultured with shaking at 28 ° C for 48 hours, and then 10.5 g of cells were collected using a centrifuge.
(2)染色体 DNAの抽出  (2) Chromosomal DNA extraction
(1)で得られた菌体のうち湿菌体 0. 31gを液体窒素により凍結した後、粉砕し、常 法により染色体 DNAを抽出した。  Of the cells obtained in (1), 0.31 g of wet cells were frozen with liquid nitrogen, crushed, and chromosomal DNA was extracted by a conventional method.
(3) FAD結合型グルコース脱水素酵素と推定される遺伝子（AO090005000449遺 伝子）のクローニング  (3) Cloning of a gene (AO090005000449 gene) presumed to be FAD-linked glucose dehydrogenase
使用する宿主としては、ァスペルギルス 'ォリゼ NS4株を使用した。本菌株は、公知 文献 1にあるように、 1997年（平成 9年）に醸造試験所で育種され、転写因子の解析、 各種酵素の高生産株の育種などに利用され、分譲されているものが入手可能である  As the host to be used, Aspergillus oryzae NS4 strain was used. This strain is bred in a brewery laboratory in 1997 as described in known literature 1, and is used for analysis of transcription factors, breeding of high-producing strains of various enzymes, etc. Is available
本菌株に対し、公知文献 2に記載してある、ァスペルギルス 'ォリゼ由来のアミラー ゼ系の改良プロモーターを使用し、その下部に（2)で得られた染色体 DNAを铸型と して、実施例 2で使用したプライマー（配列番号 8及び配列番号 9)を用いて増幅した FAD結合型グルコース脱水素酵素と推定される遺伝子（AO090005000449遺伝 子）を結合させることで、本遺伝子が発現可能なベクターを調製した。 For this strain, an improved amylase-based promoter derived from Aspergillus oryzae described in publicly known document 2 was used, and the chromosomal DNA obtained in (2) was used as a cage in the lower part. A vector capable of expressing this gene by binding the gene (AO090005000449 gene) presumed to be FAD-linked glucose dehydrogenase amplified using the primers used in 2 (SEQ ID NO: 8 and SEQ ID NO: 9) Prepared.
形質転換は、基本的には公知文献 2及び公知文献 3に記載の方法に準じて実施 することで、形質転換体を取得した。  Transformation was basically carried out according to the methods described in known literature 2 and known literature 3 to obtain transformants.
実施例 3 Example 3
(遺伝子配列の確認） (Confirmation of gene sequence)
( 1 )組換え大腸菌中のァスペルギルス ·オリゼ NBRC 5375株由来 FAD結合型グル コース脱水素酵素と推定される遺伝子の配列  (1) Aspergillus oryzae NBRC 5375 strain derived from recombinant E. coli FAD-linked glucose dehydrogenase
実施例 1で得られた組換え大腸菌中のァスペルギルス ·ォリゼ NBRC5375株由 来 FAD結合型グルコース脱水素酵素と推定される遺伝子の配列決定を行った結果 を配列番号 3に示した。配列番号 3の配列と比較例における FAD結合型グルコース 脱水素酵素と想定される遺伝子（AO090005000449)の塩基配列からイントロンを 除いた cDNA配列を比較したところ、 AO090005000449の開始塩基 Aを 1番目とし た時の 604番目力ら 606番目の ATGとレ、う配列は、ァスペルギルス.ォリゼ NBRC 5375株由来 FAD結合型グルコース脱水素酵素と推定される遺伝子では配列番号 5 に示した GCTGGTGTTCCATGGGTTという配列であり、その他の配列は完全に一致 していた。 The result of sequencing the gene presumed to be FAD-linked glucose dehydrogenase derived from Aspergillus oryzae NBRC5375 in the recombinant Escherichia coli obtained in Example 1 is shown in SEQ ID NO: 3. When comparing the sequence of SEQ ID NO: 3 with the cDNA sequence of the gene assumed to be FAD-linked glucose dehydrogenase (AO090005000449) in the comparative example, excluding the intron, the starting base A of AO090005000449 is the first. The 604th force and the 606th ATG and the retinal sequence at that time are the GCTGGTGTTCCATGGGTT sequence shown in SEQ ID NO: 5 in the gene presumed to be FAD-bound glucose dehydrogenase derived from Aspergillus oryzae NBRC 5375 strain, The other sequences were perfectly matched.
また、翻訳したアミノ酸配列を配列番号 1に示し、同様に比較したところ、 AO09000 5000449の開始アミノ酸 Mを 1番目とした時の 202番目の Mは、ァスペルギルス-ォ リゼ NBRC 5375株由来 FAD結合型ダルコース脱水素酵素と推定される遺伝子が コードするアミノ酸配列では配列番号 4に示した AGVPWVという配列であり、その他 の配列は完全に一致していた。  The translated amino acid sequence is shown in SEQ ID NO: 1 and compared in the same manner. When the starting amino acid M of AO09000 5000449 is the first, the 202nd M is the FAD-bound dalcose derived from Aspergillus oryzae NBRC 5375 strain The amino acid sequence encoded by the gene deduced to be dehydrogenase was the sequence AGVPWV shown in SEQ ID NO: 4, and the other sequences were completely identical.
(2)組換え力ビ中のァスペルギルス 'オリゼ NBRC 5375株由来 FAD結合型ダルコ ース脱水素酵素と推定される遺伝子の配列  (2) Sequence of a gene presumed to be FAD-bound dalcose dehydrogenase derived from Aspergillus oryzae NBRC 5375 in recombinant strains
実施例 2で得られた組換え力ビ中のァスペルギルス 'ォリゼ NBRC 5375株由来 F AD結合型グルコース脱水素酵素と推定される遺伝子の配列決定を行った結果を配 列番号 2に示した。配列番号 2の配列と比較例における FAD結合型グルコース脱水 素酵素と推定される遺伝子 (AO090005000449)の塩基配列を比較したところ、 A 0090005000449の開台塩基 Aを 1番目とした B寺の 656番目力ら 658番目の ATG とレ、う配列は、ァスペルギルス'ォリゼ NBRC5375株 FAD結合型グルコース脱水素 酵素と推定される遺伝子では配列番号 5に示した GCTGGTGTTCCATGGGTTという 配列だった。  The result of sequencing the gene presumed to be FAD-bound glucose dehydrogenase derived from Aspergillus olisee NBRC 5375 strain in the recombinant strain obtained in Example 2 is shown in SEQ ID NO: 2. A comparison of the sequence of SEQ ID NO: 2 and the base sequence of the gene (AO090005000449), which is presumed to be a FAD-linked glucose dehydrin enzyme in the comparative example, showed that A The 658th ATG and ras sequence was the GCTGGTGTTCCATGGGTT sequence shown in SEQ ID NO: 5 in the gene presumed to be Aspergillus oryzae NBRC5375 strain FAD-linked glucose dehydrogenase.
また、翻訳したアミノ酸配列を配列番号 1に示し、同様に比較したところ、 AO09000 5000449 (コリン脱水素酵素と推定）の開始アミノ酸 Mを 1番目とした時の 202番目 の Mは、ァスペルギルス'ォリゼ NBRC5375株由来 FAD結合型グルコース脱水素 酵素と推定される遺伝子がコードするアミノ酸配列では配列番号 4に示した AGVPW Vとレ、う配列であり、その他の配列は一致してレ、た。  The translated amino acid sequence is shown in SEQ ID NO: 1 and compared in the same manner. When the starting amino acid M of AO09000 5000449 (presumed to be choline dehydrogenase) is the first, the 202nd M is the Aspergillus oryzae NBRC5375. The amino acid sequence encoded by the gene presumed to be a strain-derived FAD-linked glucose dehydrogenase was AGVPW V shown in SEQ ID NO: 4, and the other sequences were identical.
(遺伝子配列の比較） (Gene sequence comparison)
以上の結果より、実施例 1 , 2の菌株と比較例の菌株は、 FAD結合型グルコース脱 水素酵素と推定される遺伝子において、類似の遺伝子配列を有しているが、実施例 1 , 2のァスペルギルス.ォリゼ NBRC5375株に由来する遺伝子配列は、比較例の 菌株に由来する AO090005000449の遺伝子酉己歹 IJと比較して、 656番目力ら 658 番目の ATGという配歹が、配列番号 5に示した GCTGGTGTTCCATGGGTTという配 列になっており、またアミノ酸配列で比較して、 202番目付近のアミノ酸 Mが、配列番 号 4に示した AGVPWVになっていることが判明した。 From the above results, the strains of Examples 1 and 2 and the strains of Comparative Examples have similar gene sequences in the gene presumed to be FAD-linked glucose dehydrogenase. The gene sequence derived from Aspergillus oryzae NBRC5375 Compared with the IO IJ gene of AO090005000449 derived from the strain, the arrangement of ATG at the 656th force and the 658th is the sequence GCTGGTGTTCCATGGGTT shown in SEQ ID NO: 5, and compared with the amino acid sequence. Thus, the amino acid M near the 202nd position was found to be AGVPWV shown in SEQ ID NO: 4.
実施例 4  Example 4
[0065] (遺伝子レベルでの解析と比較） [0065] (Analysis and comparison at the gene level)
(1)サザンブロッテイングによる確認、  (1) Confirmation by Southern blotting,
実施例 2および比較例で取得した菌株を元に培養した湿菌体から、定法により DNA を抽出し、本 FAD結合型グルコース脱水素酵素と想定される遺伝子の一部をプロ一 ブにしてサザンブロッテイングによる検出をした。  DNA was extracted from the wet cells cultured based on the strains obtained in Example 2 and Comparative Example by a conventional method, and a part of the gene assumed to be the FAD-bound glucose dehydrogenase was probed as a Southern. Detection was performed by blotting.
その結果、いずれの菌株においても、ァスペルギルス.ォリゼ由来のアミラーゼ系の 改良プロモーターと結合した FAD結合型グルコース脱水素酵素と想定される遺伝子 を含む DNA断片が、それぞれ同程度のコピー数含まれていることが判明した。  As a result, in both strains, DNA fragments containing the gene assumed to be FAD-linked glucose dehydrogenase linked to the improved promoter of amylase derived from Aspergillus oryzae contain the same number of copies. It has been found.
つまり、実施例 2および比較例で取得した菌株には、形質転換によりそれぞれ同程 度のコピー数の遺伝子を含んでいることが判明した。  That is, it was found that the strains obtained in Example 2 and the Comparative Example each contained the same copy number of the gene by transformation.
(2)ノーザンブロッテイングによる確認  (2) Confirmation by Northern blotting
実施例 2および比較例で取得した菌株を元に培養した湿菌体から、定法により RNA を抽出し、本 FAD結合型グルコース脱水素酵素と想定される遺伝子の一部をプロ一 ブにしてノーザンブロッテイングによる検出をした。  RNA was extracted from the wet cells cultured based on the strains obtained in Example 2 and the comparative example by a conventional method, and a part of the gene assumed to be the FAD-binding glucose dehydrogenase was probed as a northern region. Detection was performed by blotting.
その結果、いずれの菌株においても、形質転換した菌株においては、それぞれ同程 度、ァスペルギルス'オリゼ由来のアミラーゼ系の改良プロモーターと結合した FAD結 合型グノレコース脱水素酵素遺伝子のものと推定される mRNA断片が検出された。つ まり、実施例 2および比較例で取得した菌株は、本 FAD結合型グルコース脱水素酵 素と想定される遺伝子を同程度 RNAに転写していると判断できた。  As a result, in each of the strains, the transformed strain was estimated to be the same as that of the FAD-bound gnolecose dehydrogenase gene bound to the improved amylase promoter derived from Aspergillus oryzae. Fragments were detected. In other words, the strains obtained in Example 2 and Comparative Example could be judged to have transcribed the gene assumed to be the present FAD-binding glucose dehydrogenase to the same degree of RNA.
実施例 5  Example 5
[0066] (形質転換された菌株における FAD結合型グルコース脱水素酵素の活性確認） 実施例 1の菌体については、 50 i g/mLアンピシリンナトリウム及び 0. ImMイソプ ロピノレ一 β—D—1—チォガラタトピラノシド（シグマアルドリッチジャパン社製）を含む LB液体培地で、 37°Cで 17時間振とう培養し、培養終了後、集菌、 50mMリン酸カリ ゥム緩衝液 (PH7. 0)に懸濁し、超音波破砕装置を用いて菌体を破砕後、遠心して 上清を回収し、無細胞抽出液を取得した。 [0066] (Confirmation of activity of FAD-linked glucose dehydrogenase in transformed strain) Regarding the cells of Example 1, 50 ig / mL ampicillin sodium and 0. ImM isopropinole β-D-1-thio Contains Galatatopyranoside (Sigma Aldrich Japan) Incubate in LB liquid medium for 17 hours at 37 ° C. After completion of the culture, collect the cells, suspend them in 50 mM potassium phosphate buffer (PH 7.0), and remove the cells using an ultrasonic crusher. After crushing, the supernatant was collected by centrifugation, and a cell-free extract was obtained.
無細胞抽出液を SDS— PAGEに供したところ、分子量約 63kDaの酵素蛋白を確認 でき、無細胞抽出液当たり、 0. 014UZmLの FAD結合型グルコース脱水素酵素活 性を確認した。なお、宿主である大腸菌には本活性は全く認められなかった。 When the cell-free extract was subjected to SDS-PAGE, an enzyme protein having a molecular weight of about 63 kDa was confirmed, and 0.014 UZmL of FAD-linked glucose dehydrogenase activity was confirmed per cell-free extract. In addition, this activity was not recognized at all in the host E. coli.
実施例 2および比較例の菌体については、ペプトン 1。/。、ショ糖 2%、リン酸水素二力 リウム 0.5%、硫酸マグネシウム 0.05。/。を含む培養液で、 28°C、 3日間振盪培養し、培 養終了後、遠心して菌体及び培養上清を回収、菌体を 50mMリン酸カリウム緩衝液（ pH7. 0)に懸濁し、チップ式超音波破砕装置を用いて菌体を破砕後、遠心して上清 を回収し、無細胞抽出液とした。 Peptone 1 for the cells of Example 2 and Comparative Example. /. , Sucrose 2%, hydrogen phosphate 0.5%, magnesium sulfate 0.05. /. And cultured at 28 ° C for 3 days with shaking. After culturing, the cells and the culture supernatant are collected by centrifugation, and the cells are suspended in 50 mM potassium phosphate buffer (pH 7.0). The cells were crushed using a chip-type ultrasonic crusher, centrifuged, and the supernatant was collected to obtain a cell-free extract.
培養上清及び無細胞抽出液を SDS— PAGEに供したところ、実施例 2の菌体にお いては、培養上清に分子量約 86kDaの酵素蛋白を確認できた力 比較例の菌体に おいては培養上清及び無細胞抽出液にも確認ができなかった。 When the culture supernatant and the cell-free extract were subjected to SDS-PAGE, the ability of the bacterial cell of Example 2 to confirm an enzyme protein having a molecular weight of about 86 kDa was confirmed in the bacterial cell of Comparative Example. Neither the culture supernatant nor the cell-free extract could be confirmed.
また、前述の酵素活性測定法に従い、培養上清及び無細胞抽出液の FAD結合型グ ルコース脱水素酵素活性を確認したところ、実施例 2の菌体においては、培養上清 に 53U/mLの FAD結合型グルコース脱水素酵素活性を確認したが、比較例の菌 体にぉレ、ては、培養上清及び無細胞抽出液に全く活性が確認できなかった。 In addition, when the FAD-bound glucose dehydrogenase activity of the culture supernatant and the cell-free extract was confirmed according to the enzyme activity measurement method described above, in the cell of Example 2, the culture supernatant was 53 U / mL. Although the FAD-bound glucose dehydrogenase activity was confirmed, no activity was confirmed in the culture supernatant and the cell-free extract.
(まとめ） (Summary)
実施例 3〜5の知見をまとめると、実施例 2と比較例は、形質転換されている遺伝子 のコピー数およびその転写量までは同等である力 S、形質転換されている FAD結合型 グノレコース脱水素酵素の遺伝子と想定される配列が微妙に異なっており、その遺伝 子配列の違レ、が酵素活性の発現に大きな影響を与えてレ、ると結論付けることができ る。 Summarizing the findings of Examples 3-5, Example 2 and Comparative Example show that the number of copies of the transformed gene and the amount of transcription are equivalent up to the amount of transcription S. It can be concluded that the sequence assumed to be a gene of elementary enzyme is slightly different, and that the difference in the gene sequence has a great influence on the expression of enzyme activity.
実施例 6 Example 6
(ァスペルギルス.ォリゼの他の菌株における比較） (Comparison with other strains of Aspergillus oryzae)
ァスペルギルス.ォリゼの他の数種類の菌株について、実施例 1一（2)と同様にそ れらの培養上清及び無細胞抽出液（CFE)における FAD結合型グルコース脱水素酵 素活性を確認した。また、それらの菌株について、実施例 2— (1)と同様に染色体 DN Aを抽出し、配列番号 6及び 7記載のプライマーを使用し増幅した約 1. 9kbpの断片 の配列を決定し、配列番号 2に記載の配列、並びに AO090005000449の染色体 DNA配列と比較した。また、翻訳したアミノ酸配列を、配列番号 1に記載の配列、並 びに AO090005000449のアミノ酸配歹 1Jと比較した。これらの結果を、実施例 1から 3 及び比較例の結果と共に以下の表 1に示した。配列に関しては特に実施例 3— (1) 記載の AGVPWVというアミノ酸配列の有無に関して表 1に示した。 For several other strains of Aspergillus oryzae, in the same manner as in Example 1 (1) (2), FAD-linked glucose dehydrogenation in their culture supernatant and cell-free extract (CFE) Elementary activity was confirmed. For these strains, chromosome DNA was extracted in the same manner as in Example 2- (1), and the sequence of the approximately 1.9 kbp fragment amplified using the primers described in SEQ ID NOs: 6 and 7 was determined. Comparison was made with the sequence of number 2 and the chromosomal DNA sequence of AO090005000449. Further, the translated amino acid sequence was compared with the sequence shown in SEQ ID NO: 1 and the amino acid sequence 1J of AO090005000449. These results are shown in Table 1 below together with the results of Examples 1 to 3 and Comparative Example. Regarding the sequence, the presence or absence of the amino acid sequence AGVPWV described in Example 3- (1) is shown in Table 1.
[表 1] [table 1]
Figure imgf000028_0001
Figure imgf000028_0001
ァスぺノレギノレス *才リゼ NBRC4079, 4214、 4268、 5238、 6215及び 30113由 来の染色体 DNA配列は、いずれも配列番号 2の配列と完全に一致していた。  Aspenoleginores * years lyse NBRC4079, 4214, 4268, 5238, 6215 and 30113 all chromosomal DNA sequences were completely identical to the sequence of SEQ ID NO: 2.
ァスペルギルス.ォリゼ NBRC4203由来の染色体 DNA配列は、配列番号 2の配 列と 4塩基（135C→A, 437G→A, 532G→A, 1263C→T)が異なっていた。さらに、ァ スペルギルス.ォリゼ NBRC4203由来の染色体 DNA配列を翻訳したアミノ酸配列 は、配列番号 1の配列と 2アミノ酸（129V→I， 386A→V)が異なっていた。  The chromosomal DNA sequence derived from Aspergillus oryzae NBRC4203 differed from the sequence of SEQ ID NO: 2 in 4 bases (135C → A, 437G → A, 532G → A, 1263C → T). Furthermore, the amino acid sequence obtained by translating the chromosomal DNA sequence derived from Aspergillus oryzae NBRC4203 differed from the sequence of SEQ ID NO: 1 by 2 amino acids (129V → I, 386A → V).
また、ァスペルギルス'オリゼ NBRC30104由来の染色体 DNA配列は、配列番号 2の配歹 1Jと 4塩基（135C→A， 413C→A, 437G→A, 532G→A)が異なっていた。さらに 、ァスペルギルス'ォリゼ NBRC30104由来の染色体 DNA配列を翻訳したアミノ酸 配列は、配列番号 1の配列と 2アミノ酸（121R→S, 129V→I)が異なっていた。これら のアミノ酸配列の違いは、 FAD結合型グルコース脱水素酵素の発現には直接影響し ていないものと推察された。 Further, the chromosomal DNA sequence derived from Aspergillus oryzae NBRC30104 was different in the arrangement 1J of SEQ ID NO: 2 and 4 bases (135C → A, 413C → A, 437G → A, 532G → A). Furthermore, translated amino acid sequence of chromosomal DNA derived from Aspergillus oryzae NBRC30104 The sequence was different from the sequence of SEQ ID NO: 1 by 2 amino acids (121R → S, 129V → I). It was speculated that these amino acid sequence differences did not directly affect the expression of FAD-bound glucose dehydrogenase.
また、ァスペルギルス'オリゼ NBRC4181及び 4220の染色体 DNA配列は、レヽ ずれも AO090005000449の染色体 DNA酉己歹 IJと完全に一致してレ、た。  Furthermore, the chromosomal DNA sequences of Aspergillus oryzae NBRC4181 and 4220 were completely consistent with the chromosomal DNA IJ of AO090005000449.
実施例:!〜 5及び比較例の結果より、ァスペルギルス'オリゼ NBRC5375株由来の FAD結合型グルコース脱水素酵素と推定される遺伝子は、活性型の FAD結合型グ ルコース脱水素酵素をコードする遺伝子だったと結論付けられ、また、ァスペルギル ス-ォリゼ NBRC100959株由来 FAD結合型グノレコース脱水素酵素と推定される遺伝 子（AO090005000449遺伝子）は、活性型の FAD結合型グルコース脱水素酵素を コードする遺伝子ではなかった。 AO090005000449遺伝子は、 NBRC5375株由 来の FAD結合型グノレコース脱水素酵素のアミノ酸配列と非常に類似したアミノ酸配 列をコードしているので、当該技術分野における技術常識に鑑みると、同様の酵素 活性を持っていると考えられる。し力しながら、予想外なことに、本発明者によって、 実際に ίま匕較列に示すように AO090005000449遺伝子、 NBRC4181遺伝子及 び 4220遺伝子の配列では該酵素は発現されないことが初めて見出された。同様の 発現系を用いて、前記の配列の違いのみで FAD結合型グルコース脱水素酵素の発 現の有無が生じていることから、あくまで仮説ではある力 ァスペルギルス 'ォリゼ N BRC 5375株等の FAD結合型ダルコース脱水素酵素に含まれるアミノ酸配列： AGVP WVは FAD結合型グルコース脱水素酵素の高次構造を取るのに重要な配列と思わ れ、 AGVPWVが欠けた場合は小胞体ストレス等を引き起こし、発現蛋白の分解及び /または発現の抑制が起こっていると推察される。開始アミノ酸 Mを 1番目とした時に 202番目付近にアミノ酸配列: AGVPWVが存在することは機能発現に重要である。な おこの配列中のどのアミノ酸が活性発現に必須であるかは、現在研究中である力 一 部のアミノ酸を欠失、置換または付加してもある程度の活性は維持できる可能性はあ る。また、アミノ酸配列： AGVPWV以外の部分においては、ァスペルギルス'ォリゼ N BRC4203,ァスペルギルス'オリゼ NBRC 30104の遺伝子解析から判明した数個 のアミノ酸置換は、 FAD結合型グルコース脱水素酵素の発現に影響を与えなかった 実施例 7 Examples: From the results of! ~ 5 and the comparative example, the gene presumed to be FAD-bound glucose dehydrogenase derived from Aspergillus oryzae NBRC5375 is a gene encoding an active FAD-bound glucose dehydrogenase. In addition, the gene presumed to be FAD-linked gonorlecose dehydrogenase derived from Aspergillus oryzae NBRC100959 (AO090005000449 gene) was not a gene encoding active FAD-linked glucose dehydrogenase. . The AO090005000449 gene encodes an amino acid sequence that is very similar to the amino acid sequence of the FAD-bound gnolecose dehydrogenase derived from the NBRC5375 strain. It is thought that. However, unexpectedly, the present inventor found for the first time that the enzyme is not expressed in the sequences of the AO090005000449 gene, the NBRC4181 gene, and the 4220 gene as shown in the comparison column. It was. Using the same expression system, the presence or absence of the expression of FAD-bound glucose dehydrogenase is caused only by the difference in the above-mentioned sequences. Therefore, the hypothesis is that the strength of FAD binding of Aspergillus oryzae N BRC 5375 Amino acid sequence contained in type dulcose dehydrogenase: AGVP WV appears to be an important sequence for adopting the higher-order structure of FAD-linked glucose dehydrogenase. If AGVPWV is lacking, it is expressed by causing endoplasmic reticulum stress. It is inferred that protein degradation and / or suppression of expression occurs. It is important for functional expression that the amino acid sequence: AGVPWV exists near the 202nd position when the starting amino acid M is the first position. It should be noted that which amino acids in this sequence are essential for the expression of activity can be maintained to some extent even if some of the amino acids currently under study are deleted, substituted or added. In addition to the amino acid sequence: AGVPWV, several amino acid substitutions revealed by gene analysis of Aspergillus olirise N BRC4203 and Aspergillus olisee NBRC 30104 did not affect the expression of FAD-bound glucose dehydrogenase. The Example 7
[0071] (FAD結合型グルコース脱水素酵素の性質試験）  [0071] (Property test of FAD-linked glucose dehydrogenase)
実施例 5で得られた、実施例 2の菌体の培養上清を、分画分子量 1万のビバセル 2 (ビバサイエンス社製）で濃縮後、蒸留水で置換し、タンパク質当たりの比活性が 323 UZmgの精製酵素を得た。尚、 FAD結合型グルコース脱水素酵素活性を示した他 の菌株由来の酵素についても、同様に精製可能であった。これらの精製酵素を SDS — PAGEに供したところ、約 86kDaの単一バンドを確認できた。本酵素について、作 用性、基質特異性及び補酵素を調べた。なお、酵素活性は、前述記載の酵素活性 測定法に従って測定した。  The culture supernatant of the cells of Example 2 obtained in Example 5 was concentrated with Vivacel 2 (manufactured by Viva Science) with a molecular weight cut-off of 10,000, and then replaced with distilled water. 323 UZmg of purified enzyme was obtained. Enzymes derived from other strains that showed FAD-linked glucose dehydrogenase activity could be purified in the same manner. When these purified enzymes were subjected to SDS-PAGE, a single band of about 86 kDa was confirmed. For this enzyme, the operability, substrate specificity and coenzyme were examined. The enzyme activity was measured according to the enzyme activity measurement method described above.
1)作用性  1) Action
精製酵素を、 8. 66mM DCIP存在下で 500mM D—グルコースと反応させ、反 応産物を D—ダルコン酸/ D—ダルコノー δ—ラタトン定量キットで定量した。その結 果、 D—ダルコン酸の生成が確認され、これより本発明の FAD結合型グルコース脱水 素酵素は D—グノレコースの 1位の水酸基を酸化する反応を触媒する酵素であること が明らかになった。  The purified enzyme was reacted with 500 mM D-glucose in the presence of 8.66 mM DCIP, and the reaction product was quantified with the D-Dalconic acid / D-Dalcono δ-Lataton quantification kit. As a result, the production of D-dalconic acid was confirmed, and it was clarified from this that the FAD-bound glucose dehydrating enzyme of the present invention is an enzyme that catalyzes the reaction of oxidizing the hydroxyl group at the 1-position of D-gnolecose. It was.
2)基質特異性  2) Substrate specificity
前述の酵素活性測定法における活性測定用反応液の基質を、 D—グルコース、マ ノレトース、及び D—ガラ外ースを使用し、酵素活性測定法に則り精製酵素の酵素活 性を測定した。該酵素の D—グノレコースに対する活性値を 100%とした場合、マルト ースに対する酵素活性値が 2. 1%、 D—ガラクトースに対する酵素活性値が 0. 99% の作用性だった。  The enzyme activity of the purified enzyme was measured according to the enzyme activity measurement method, using D-glucose, maleoleose, and D-galactose as the substrate for the activity measurement reaction solution in the enzyme activity measurement method described above. When the activity value of the enzyme for D-gnolecose was 100%, the enzyme activity value for maltose was 2.1%, and the enzyme activity value for D-galactose was 0.99%.
3)補酵素  3) Coenzyme
精製酵素に D—グノレコースを添カ卩し、吸光分析を行ったところ、 385nm及び 465η mに認められた吸収極大が該添加により消失したことから、補酵素が FADであること が明らかとなった。  When D-gnolecose was added to the purified enzyme and an absorption analysis was performed, the absorption maxima observed at 385 nm and 465 ηm disappeared by the addition, indicating that the coenzyme was FAD. .
実施例 8  Example 8
[0072] (酵素固定化電極によるグルコースの測定） 実施例 7記載の精製酵素を使用し、酵素固定化電極による D—グルコースの測定 を行った。本酵素 1. 5Uを固定化したグラッシ一カーボン（GC)電極を用いて、ダル コース濃度に対する応答電流値を測定した。電解セル中に、 50mM リン酸カリウム 緩衝液（PH6. 0) 1. 8ml及び 1M へキサシァノ鉄（III)酸カリウム（フェリシアン化力 リウム）水溶液 0. 2mlを添加した。 GC電極をポテンシヨスタツト BAS100BZW (BA S製）に接続し、 37°Cで溶液を撹拌し、銀塩ィ匕銀参照電極に対して + 500mVを印 カロした。これらの系に 1M D—グルコース溶液を終濃度が 5、 10、 20、 30、 40、 50 mMになるよう添加し、添加ごとに定常状態の電流値を測定した。この電流値を既知 のグノレコース濃度（5、 10、 20、 30、 40、 50mM)に対してプロットしたところ、検量線 が作成できた（図 1)。これより本発明の FAD結合型グルコース脱水素酵素を使用した 酵素固定化電極でグノレコースの定量が可能であることが示された。 [0072] (Measurement of glucose with enzyme-immobilized electrode) Using the purified enzyme described in Example 7, D-glucose was measured with an enzyme-immobilized electrode. Using a glassy carbon (GC) electrode on which 1.5 U of this enzyme was immobilized, the response current value for the dalcose concentration was measured. In the electrolysis cell, 1.8 ml of 50 mM potassium phosphate buffer (PH 6.0) and 0.2 ml of 1 M potassium hexanoferrate (III) potassium ferricyanide solution were added. The GC electrode was connected to a potentiostat BAS100BZW (manufactured by BAS), the solution was stirred at 37 ° C., and +500 mV was printed on the silver salt / silver reference electrode. A 1M D-glucose solution was added to these systems to a final concentration of 5, 10, 20, 30, 40, and 50 mM, and a steady-state current value was measured for each addition. When this current value was plotted against the known gnolecose concentration (5, 10, 20, 30, 40, 50 mM), a calibration curve was created (Fig. 1). From this, it was shown that quantorecose can be quantified with an enzyme-immobilized electrode using the FAD-linked glucose dehydrogenase of the present invention.
実施例 9  Example 9
[0073] (PCRによる FAD結合型グルコース脱水素酵素遺伝子の確認）  [0073] (Confirmation of FAD-linked glucose dehydrogenase gene by PCR)
(1)菌体培養  (1) Cell culture
グルコース (ナカライ社製） 1 % (W/V)、脱脂大豆 (昭和産業社製) 2% (W/V)、コ ーンスティープリカ一（サンエイ糖化社製） 0. 5% (W/V)、硫酸マグネシウム七水和 物（ナカライネ土製） 0. 1 % (WZV)及び水からなる液体培地を pH6. 0に調整し、 10 mLを太試験管に入れ、 121°C、 20分間オートクレープした。冷却したこの液体培地 に、実施例 4で示しているように、培養液中にグルコース脱水素酵素活性を有するこ とが確認されているァスペルギルス'ォリゼ NBRC4268株、 NBRC5375株、 NBRC6215 株と、培養液中に本酵素活性が認められないァスペルギルス'ォリゼ NBRC4181株、 NBRC4220株、及び NBRC100959株を各試験管に接種し、 30°Cで 43時間振盪培養 した後、遠心分離機を用いて、それぞれ湿菌体を回収した。  Glucose (Nacalai) 1% (W / V), defatted soybean (Showa Sangyo) 2% (W / V), Cone steep liquor (SUNEI KAGAKU) 0.5% (W / V ), Magnesium sulfate heptahydrate (Nacalaine) 0.1% (WZV) and a liquid medium consisting of water are adjusted to pH 6.0, 10 mL is placed in a thick test tube, and autoclaved at 121 ° C for 20 minutes. did. As shown in Example 4, in this cooled liquid medium, Aspergillus oryzae NBRC4268 strain, NBRC5375 strain, NBRC6215 strain, which have been confirmed to have glucose dehydrogenase activity in the culture broth, and the culture broth Aspergillus oryzae NBRC4181 strain, NBRC4220 strain, and NBRC100959 strain, in which this enzyme activity is not observed, were inoculated into each test tube, cultured at 30 ° C for 43 hours with shaking, and then each was treated with a wet centrifuge using a centrifuge. The body was recovered.
[0074] (2)染色体 DNAの抽出 [0074] (2) Chromosomal DNA extraction
(1)で得られた湿菌体を液体窒素で凍結した後、粉砕し、常法により染色体 DNAを 抽出した。  The wet cells obtained in (1) were frozen in liquid nitrogen, crushed, and chromosomal DNA was extracted by a conventional method.
[0075] (3) FAD結合型グルコース脱水素酵素遺伝子全長の増幅  [0075] (3) Amplification of the full-length FAD-linked glucose dehydrogenase gene
(2)で抽出した各 DNAをテンプレートに、配列番号 2の配列を元に合成したプライマ 一 3及び 4を用いて、下記条件で PCRを行い、約 1.9kbpの FAD結合型グルコース脱 水素酵素遺伝子を含む PCR産物を取得した。 Primers synthesized based on the sequence of SEQ ID NO: 2 using each DNA extracted in (2) as a template PCR was carried out using 1 and 3 under the following conditions to obtain a PCR product containing an approximately 1.9 kbp FAD-linked glucose dehydrogenase gene.
テンプレート ：（2)で抽出した DNA  Template: DNA extracted in (2)
プライマー ：  Primer :
プライマー 3 ： 5 ' -ttatgctcttctcactggcattcctgagtgccctgt-3 ' (酉己歹 lj番号 10)  Primer 3: 5 '-ttatgctcttctcactggcattcctgagtgccctgt-3' (酉 己 歹 lj number 10)
フフイマ" ~ 4 ： 5 -gctaagcactcttcgcatcctccttaatcaagtcgg-3 (酉己歹 'J番号 11ノ 反応条件 ：変性 94°C、 1分（1サイクル）  Huhuima "~ 4: 5 -gctaagcactcttcgcatcctccttaatcaagtcgg-3 (酉 酉 歹 J number 11) Reaction condition: Denaturation 94 ° C, 1 minute (1 cycle)
変性 94°C、 30秒、アニーリング 45°C、 30秒、伸長反応 72°C、 1分 30秒（30サイクル） 伸長反応 72°C、 10分（1サイクル）  Denaturation 94 ° C, 30 seconds, annealing 45 ° C, 30 seconds, extension reaction 72 ° C, 1 minute 30 seconds (30 cycles) Extension reaction 72 ° C, 10 minutes (1 cycle)
[0076] (4)活性を発現する FAD結合型グルコース脱水素酵素遺伝子の増幅 [0076] (4) Amplification of FAD-binding glucose dehydrogenase gene expressing the activity
(1)で得られた各 PCR産物をテンプレートに、プライマー 3とアミノ酸配列： AGVPWV を元に合成したプライマー 5を用いて、下記条件で PCRを行った。  Using each PCR product obtained in (1) as a template, PCR was performed under the following conditions using primer 3 and primer 5 synthesized based on the amino acid sequence: AGVPWV.
テンプレート ：（3)で得られた PCR産物  Template: PCR product obtained in (3)
プライマー ：  Primer :
プライマー 3 ： 5' -ttatgctcttctcactggcattcctgagtgccctgt- 3， (酉己歹 IJ番号 10) プフイマ一 5 ： 5 -aacccatggaacaccagc-3 ' (酉己列番 12)  Primer 3 ： 5 '-ttatgctcttctcactggcattcctgagtgccctgt-3, (酉 己 歹 IJ number 10) Phuima 1 5 ： 5 -aacccatggaacaccagc-3 ′ (酉 self column number 12)
反応条件 ：変性 94°C、 1分（1サイクル）  Reaction conditions: Denaturation 94 ° C, 1 minute (1 cycle)
変性 94°C、 30秒、アニーリング 65°C、 30秒、伸長反応 72°C、 1分（30サイクル） 伸長反応 72°C、 5分（1サイクル)。  Denaturation 94 ° C, 30 seconds, annealing 65 ° C, 30 seconds, extension reaction 72 ° C, 1 minute (30 cycles) Extension reaction 72 ° C, 5 minutes (1 cycle).
[0077] PCRによる目的遺伝子の検出結果を第 2図に示した。培養液中にグルコース脱水素 酵素活性を有するァスペルギルス 'ォリゼ由来の FAD結合型グルコース脱水素酵素 をコードするポリヌクレオチドのみ力 PCRで予想されるサイズの増幅を確認できた。 尚、（2)で得られた DNAを直接テンプレートに用いて PCRを行っても、同様に、培養 液中にグルコース脱水素酵素活性を有するァスペルギルス ·ォリゼ由来の FAD結合 型グノレコース脱水素酵素をコードするポリヌクレオチドのみが、 PCRで予想されるサイ ズの増幅を確認できた。 [0077] FIG. 2 shows the results of detection of the target gene by PCR. Only the polynucleotide encoding the FAD-linked glucose dehydrogenase derived from Aspergillus oryzae having glucose dehydrogenase activity in the culture medium was confirmed to have the amplification of the size expected by force PCR. In addition, even when PCR is carried out using the DNA obtained in (2) directly as a template, the FAD-linked gnorolecose dehydrogenase derived from Aspergillus oryzae having glucose dehydrogenase activity in the culture medium is similarly encoded. Only those polynucleotides that were able to confirm the amplification of the size expected by PCR.
実施例 10  Example 10
[0078] (サザンハイブリダィゼーシヨンによる FAD結合型グノレコース脱水素酵素遺伝子の確 実施例 10の（1)で得られた各 PCR産物 lOOngをァガロースゲル電気泳動後、ナイ口 ンメンブレン（Hybond-N+、 GEヘルスケア社製）にブロッテイングし、 80°Cで 71時間固 定した。プレハイブリダィゼーシヨンした後、 5 '末端をフルォレセインイソチオシァネー ト（FITC)で蛍光標識した、アミノ酸配列: AGVPWVを元に合成したプローブをカロえ、 37。Cで 24時間インキュベートした。メンブレンを 4。C、 6 X SSC及び 50°C、塩化テトラメ チルアンモニゥム溶液で洗浄後、 25mM TBSで塩ィ匕テトラメチルアンモニゥム溶液由 来の SDSを洗浄し、イメージアナライザー（Typhoon9400、 GEヘルスケア社製）で、蛍 光検出を行った。以下に、使用したバッファーの組成及びプローブの配列について 記載する。 [0078] (Confirmation of FAD-linked gnolecose dehydrogenase gene by Southern hybridization) Each PCR product lOOng obtained in (1) of Example 10 was subjected to agarose gel electrophoresis, blotted on a nylon membrane (Hybond-N +, manufactured by GE Healthcare), and fixed at 80 ° C for 71 hours. . After prehybridization, the 5 ′ end was fluorescently labeled with fluorescein isothiocyanate (FITC), and the probe synthesized based on the amino acid sequence: AGVPWV was prepared. Incubated for 24 hours at C. Membrane 4. After washing with C, 6 X SSC and 50 ° C, tetramethylammonium chloride solution, the SDS from the salty tetramethylammonium solution was washed with 25 mM TBS, and image analyzer (Typhoon9400, manufactured by GE Healthcare) was used. Fluorescence detection was performed. The buffer composition and probe sequence used are described below.
ハイブリダィゼーシヨンバッファー ： Hybridization buffer:
6 X SSC 6 X SSC
5 Xデンハルト溶液  5 X Denhardt's solution
0.5%スキムミノレク  0.5% Skim Minorek
20 X SSC ：  20 X SSC:
3M塩化ナトリウム  3M sodium chloride
0.3Mクェン酸三ナトリウム  0.3M trisodium citrate
塩化テトラメチルアンモニゥム溶液 ： Tetramethylammonium chloride solution:
3M塩化テトラメチルアンモニゥム  3M tetramethylammonium chloride
50mM Tris-HCl (pH8.0)  50 mM Tris-HCl (pH 8.0)
2mM EDTA  2mM EDTA
0.1 % SDS  0.1% SDS
プローブ ：5， (FITC) -gctggtgttccatgggtt-3 ' (配列番号 5)。 Probe: 5, (FITC) -gctggtgttccatgggtt-3 '(SEQ ID NO: 5).
サザンハイブリダィゼーシヨンによる目的遺伝子の検出結果を第 3図に示す。培養液 中にグルコース脱水素酵素を有するァスペルギルス 'オリゼ由来の FAD結合型グノレ コース脱水素酵素をコードするポリヌクレオチドのみが、サザンハイブリダィゼーシヨン で検出できることがわかる。尚、サザンハイブリダィゼーシヨンによる確認は、実施例 9 の（3)で得られた PCR産物をナイロンメンブレン（Hybond_N+、 GEヘルスケア社製）上 に固定して行っても同様の結果を得ることができる。 Fig. 3 shows the results of detection of the target gene by Southern hybridization. It can be seen that only the polynucleotide encoding the FAD-linked gnolecose dehydrogenase derived from Aspergillus oryzae having glucose dehydrogenase in the culture medium can be detected by Southern hybridization. For confirmation by Southern hybridization, the PCR product obtained in (9) of Example 9 was placed on a nylon membrane (Hybond_N +, manufactured by GE Healthcare). The same result can be obtained even if it is fixed to the above.
実施例 11  Example 11
[0080] (FAD結合型グルコース脱水素酵素遺伝子と確認できた遺伝子のクローニング、及 びクローユングした菌株における分泌生産）  [0080] (Cloning of a gene identified as a FAD-linked glucose dehydrogenase gene, and secretory production in a cloned strain)
実施例 9及び/又は実施例 10に示す方法で、培養液中にグルコース脱水素酵素を 分泌生産するァスペルギルス 'オリゼ由来の FAD結合型グノレコース脱水素酵素をコ ードするポリヌクレオチドと確認できた同遺伝子について、実施例 2に記載の方法従 つてベクターに連結、クローユングした菌株を培養した結果、培養液上清に活性型酵 素を大量に分泌生産させることができた。  It was confirmed by the method shown in Example 9 and / or Example 10 that the polynucleotide was able to be confirmed as a polynucleotide encoding an FAD-linked gnolecose dehydrogenase derived from Aspergillus oryzae that secretes and produces glucose dehydrogenase in the culture medium. As a result of culturing strains that were linked to a vector and cloned according to the method described in Example 2, the gene was secreted and produced in large quantities in the culture supernatant.
実施例 12  Example 12
[0081] (ァスペルギルス'オリゼ由来 FAD結合型グルコース脱水素酵素の活性発現に影響 を及ぼしてレ、るアミノ酸の確認）  [0081] (Confirmation of amino acids that affect the expression of FAD-linked glucose dehydrogenase derived from Aspergillus oryzae)
ァスペルギルス 'ォリゼ NBRC5375株由来 FAD結合型グルコース脱水素酵素の 6アミ ノ酸 (AGVPWV (202〜207番目のアミノ酸））のうち、 1アミノ酸を欠失させた幾つかの 変異酵素遺伝子、 6個すベてのアミノ酸を欠失させた変異酵素遺伝子、及び、それら 6個のアミノ酸に代えて Metをコードする塩基を有する変異酵素遺伝子を作成し、ァ スペルギルス ·オリゼ NS4株に導入して活性発現への影響を確認した。尚、変異遺伝 子の作成は、 STRATAGEN社製 Quik Change Site Directed Muntagenesis Kitsを 用レ、、ァスペルギルス 'ォリゼへの遺伝子導入は実施例 2に記載の方法に順じて行つ た。各々の変異導入組み換え体（単コピーと推定されるもの）にっき培地当たりの平 均活性値（3株）を求めた結果を表 2に示す。これら結果から、ァスペルギルス *ォリゼ の FAD結合型グノレコース脱水素酵素の活性発現には、これらの 6アミノ酸、特に、 205 〜207番目のアミノ酸が重要であることが強く示唆される。  Among the 6 amino acids (AGVPWV (amino acids 202 to 207)) of FAD-linked glucose dehydrogenase derived from Aspergillus olisee NBRC5375, several mutant enzyme genes lacking one amino acid, A mutant enzyme gene with all amino acids deleted, and a mutant enzyme gene having a base encoding Met in place of these 6 amino acids, were introduced into the Aspergillus oryzae NS4 strain for active expression. The effect was confirmed. The mutant gene was prepared using Quik Change Site Directed Muntagenesis Kits manufactured by STRATAGEN, and the gene transfer into Aspergillus olise was performed according to the method described in Example 2. Table 2 shows the results of calculating the average activity (3 strains) per culture medium for each mutagenized recombinant (presumed to be a single copy). These results strongly suggest that these 6 amino acids, particularly the 205th to 207th amino acids, are important for the expression of the activity of Aspergillus * olise FAD-linked gnolecose dehydrogenase.
[0082] [表 2] 変異箇所 活性値 Control (No. 1)を 100とした場合の[0082] [Table 2] Mutation location Activity value When Control (No. 1) is 100
No. (欠失アミノ酸） (U/ml) 相対活性 （％) No. (deleted amino acid) (U / ml) Relative activity (%)
1 なし (Control) 42 100 1 None (Control) 42 100
2 205 (Pro) 0. 04 0. 1以下 2 205 (Pro) 0. 04 0. 1 or less
3 206 (Trp) 0. 03 0. 1以下 3 206 (Trp) 0. 03 0. 1 or less
4 207 (Val) 0. 04 0. 1以下 4 207 (Val) 0. 04 0. 1 or less
5 202-207 0. 02 0. 1以下 5 202-207 0. 02 0. 1 or less
(Ala-Gly-Val-Pro-Trp-Val)  (Ala-Gly-Val-Pro-Trp-Val)
6 No. 8の欠失箇所に、 6アミノ酸 0. 03 0. 1以下 6 No more than amino acid 0.03 0. 1 at the deletion position of No. 8.
の代わりに Metを付加  Add Met instead of
(【比較例】 の ΑΟΟΘΟΟΟδΟΟΟ449 (Comparative example of ΘΟΟΟδΟΟΟ 449
遺伝子に相当）  Equivalent to gene)
産業上の利用可能性 Industrial applicability
本発明のポリヌクレオチドにコードされる FAD結合型グノレコース脱水素酵素は、血糖 の測定において実質的にマルトースに作用しないことから、より高精度な自己血糖測 定 (SMBG)装置にも利用することができ、糖尿病患者の自己管理 ·治療に大きく資 する。 Since the FAD-conjugated gnolecose dehydrogenase encoded by the polynucleotide of the present invention does not substantially act on maltose in the measurement of blood glucose, it can be used for a more accurate self blood glucose measurement (SMBG) apparatus. It can greatly contribute to the self-management and treatment of diabetic patients.

Claims

請求の範囲 The scope of the claims
[1] アミノ酸配列： XI- X2- X3-X4-X5-X6 [1] Amino acid sequence: XI- X2- X3-X4-X5-X6
(XI及び X2は脂肪族アミノ酸、 X3及び X6は分岐アミノ酸、並びに、 X4及び X5は複 素環式アミノ酸又は芳香族アミノ酸を示す）を含むポリペプチドから成る FAD結合型 グノレコース脱水素酵素をコードするポリヌクレオチド。  (XI and X2 are aliphatic amino acids, X3 and X6 are branched amino acids, and X4 and X5 are polycyclic amino acids or aromatic amino acids). Polynucleotide.
[2] アミノ酸配列： X1-X2-X3-X4-X5-X6が該ポリペプチドの 202〜207番目に位置する [2] Amino acid sequence: X1-X2-X3-X4-X5-X6 is located at position 202-207 of the polypeptide
、請求項 1記載のポリヌクレオチド。 The polynucleotide of claim 1.
[3] XIがァラニン (A)、 X2がグリシン（G)、 X3がバリン（V)、 X4がプロリン（P)、 X5がトリプト ファン (W)、又は、 X6がバリン (V)である、請求項 1又は 2記載のポリヌクレオチド。 [3] XI is alanine (A), X2 is glycine (G), X3 is valine (V), X4 is proline (P), X5 is tryptophan (W), or X6 is valine (V). The polynucleotide according to claim 1 or 2.
[4] アミノ酸配列： X1-X2-X3-X4-X5-X6が AGVPWVである、請求項 1記載のポリヌクレオ チド。 [4] The polynucleotide according to claim 1, wherein the amino acid sequence X1-X2-X3-X4-X5-X6 is AGVPWV.
[5] アミノ酸配列： AGVPWVから成るポリペプチドをコードする塩基配列が（GCTGGTGT [5] Amino acid sequence: The base sequence encoding the polypeptide consisting of AGVPWV is (GCTGGTGT
TCCATGGGTT)である、請求項 4記載のポリヌクレオチド。 The polynucleotide according to claim 4, which is TCCATGGGTT).
[6] ァスペルギルス'ォリゼ（Aspergillus oryzae)由来である、請求項 1ないし 5記載のポリ ヌクレオチド。 [6] The polynucleotide according to any one of claims 1 to 5, which is derived from Aspergillus oryzae.
[7] ァスペルギルス.ォリゼ（Aspergillus oryzae) NBRC5375株由来の請求項 6記載のポ リヌクレオチド。  [7] The polynucleotide according to claim 6, derived from Aspergillus oryzae NBRC5375 strain.
[8] 以下の（a)、（b)又は（c)のポリペプチドをコードするポリヌクレオチド：  [8] A polynucleotide encoding a polypeptide of the following (a), (b) or (c):
(a)配列番号 1に示されるアミノ酸配列から成るポリペプチド、  (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b)アミノ酸配列（a)のアミノ酸配列において、 1個〜数個のアミノ酸が置換、欠失又 は付加されたアミノ酸配列から成り、 FAD結合型グルコース脱水素酵素活性を有する ポリペプチド、又は  (b) a polypeptide having an FAD-linked glucose dehydrogenase activity consisting of an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence of amino acid sequence (a), or
(c)アミノ酸配列（a)と 70%以上の相同性を有するアミノ酸配列力 成り、かつ、 FAD 結合型グルコース脱水素酵素活性を有するポリペプチド。  (c) A polypeptide comprising an amino acid sequence having 70% or more homology with the amino acid sequence (a) and having FAD-bound glucose dehydrogenase activity.
[9] (b)又は（c)のポリペプチドがアミノ酸配列： X1-X2-X3-X4-X5-X6を含む、請求項 8 記載のヌクレオチド。  [9] The nucleotide according to claim 8, wherein the polypeptide of (b) or (c) comprises the amino acid sequence: X1-X2-X3-X4-X5-X6.
[10] 以下の（d)、（e)又は（f)のポリヌクレオチド： [10] The following polynucleotide (d), (e) or (f):
(d)配列番号 2又は配列番号 3に示される塩基配列を含むポリヌクレオチド、 (e)塩基配列（d)から成るポリヌクレオチドと相補的な塩基配列からなるポリヌクレオ チドとストリンジェントな条件下でハイブリダィズし、かつ、 FAD結合型グルコース脱水 素酵素活性を有するポリペプチドをコードするポリヌクレオチド、又は (d) a polynucleotide comprising the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, (e) a polynucleotide encoding a polypeptide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence (d) under stringent conditions and has a FAD-linked glucose dehydrase activity. Nucleotides, or
(f)塩基配列（d)から成るポリヌクレオチドと 70%以上の相同性を有する塩基配列を 含み、かつ、 FAD結合型グルコース脱水素酵素活性を有するポリペプチドをコードす るポリヌクレオチド。  (f) A polynucleotide encoding a polypeptide comprising a nucleotide sequence having 70% or more homology with the polynucleotide comprising the nucleotide sequence (d) and having FAD-linked glucose dehydrogenase activity.
[11] (e)又は（f)のポリヌクレオチドがアミノ酸配列：X1_X2-X3-X4-X5_X6をコードする塩 基配列を含む、請求項 10記載のポリヌクレオチド。  [11] The polynucleotide according to claim 10, wherein the polynucleotide of (e) or (f) comprises a base sequence encoding the amino acid sequence: X1_X2-X3-X4-X5_X6.
[12] アミノ酸配列： AGVPWVをコードする塩基配列力、ら成るセンスプライマー及びァスぺ ノレギルス.ォリゼ（Aspergillus oryzae)由来の FAD結合型グルコース脱水素酵素をコ ードするポリヌクレオチドの 3 '末端側の塩基配列から成るリバースプライマー、又は 、アミノ酸配列： AGVPWVをコードする塩基配列対するアンチセンスプライマー及び ァスペルギルス'ォリゼ（Aspergillus oryzae)由来の FAD結合型グルコース脱水素酵 素をコードするポリヌクレオチドの 5'末端側の塩基配列から成るフォワードプライマ 一の組み合わせを用いる PCRによって増幅可能な DNA断片を有する、 FAD結合型 グノレコース脱水素酵素活性を有するポリペプチドをコードするポリヌクレオチド。  [12] Amino acid sequence: nucleotide sequence coding for AGVPWV, sense primer and 3 'terminal side of polynucleotide encoding FAD-linked glucose dehydrogenase derived from Aspergillus oryzae 5 'terminal of a polynucleotide encoding a FAD-linked glucose dehydrogenase derived from a reverse primer comprising the nucleotide sequence of the above, or an antisense primer corresponding to the nucleotide sequence encoding the amino acid sequence AGVPWV and Aspergillus oryzae A polynucleotide encoding a polypeptide having FAD-linked gno-recose dehydrogenase activity, which has a DNA fragment that can be amplified by PCR using a combination of forward primers consisting of a base sequence on the side.
[13] アミノ酸配列： AGVPWVをコードする塩基配列力 成るプローブとストリンジヱントな条 件下でハイブリダィズし、かつ、 FAD結合型グノレコース脱水素酵素活性を有するポリ ペプチドをコードするポリヌクレオチド。  [13] Amino acid sequence: A polynucleotide encoding a polypeptide that hybridizes under stringent conditions with a probe that has the ability to sequence AGVPWV, and that has FAD-linked gnolecose dehydrogenase activity.
[14] アミノ酸配列： AGVPWVをコードする塩基配列が（GCTGGTGTTCCATGGGTT)であ る、請求項 12又は 13記載のポリヌクレオチド。  [14] The amino acid sequence: The polynucleotide according to claim 12 or 13, wherein the base sequence encoding AGVPWV is (GCTGGTGTTCCATGGGTT).
[15] D_グルコースに対する酵素活性値を 100%とした場合、マルトースに対する酵素活 性値が 10%以下、 D _ガラクトースに対する酵素活性値が 5 %以下であることを特徴 とする、ァスペルギルス'ォリゼ（Aspergillus oryzae)由来の FAD結合型グルコース脱 水素酵素をコードするポリヌクレオチド。  [15] When the enzyme activity value for D_glucose is 100%, the enzyme activity value for maltose is 10% or less, and the enzyme activity value for D_galactose is 5% or less. A polynucleotide encoding a FAD-linked glucose dehydrogenase derived from (Aspergillus oryzae).
[16] 300U/mg以上の酵素活性を有することを特徴とする、ァスペルギルス 'ォリゼ (Asp ergillus oryzae)由来の FAD結合型グルコース脱水素酵素をコードするポリヌクレオチ ド'。 [16] A polynucleotide encoding a FAD-linked glucose dehydrogenase derived from Aspergillus oryzae, characterized by having an enzyme activity of 300 U / mg or more.
[17] 請求項 1ないし 16記載のポリヌクレオチドを保有する組換えベクター。 [17] A recombinant vector comprising the polynucleotide according to any one of claims 1 to 16.
[18] 請求項 17記載の組換えベクターを用いることによって作成された形質転換細胞。  [18] A transformed cell produced by using the recombinant vector according to claim 17.
[19] 大腸菌又はァスペルギルス ·ォリゼである、請求項 18記載の形質転換細胞。  [19] The transformed cell according to claim 18, which is Escherichia coli or Aspergillus oryzae.
[20] 請求項 18又は 19記載の形質転換細胞を培養し、得られた培養物から、グルコース を脱水素する作用を有する FAD結合型グルコース脱水素酵素を採取することを特徴 とする FAD結合型グルコース脱水素酵素の製造方法。  [20] The FAD-binding glucose dehydrogenase having an action of dehydrogenating glucose is collected from the obtained culture by culturing the transformed cell according to claim 18 or 19. A method for producing glucose dehydrogenase.
[21] 請求項 1ないし 16記載のポリヌクレオチドにコードされる、組換え FAD結合型ダルコ ース脱水素酵素。 [21] A recombinant FAD-bound dalcose dehydrogenase encoded by the polynucleotide according to any one of claims 1 to 16.
[22] 請求項 21に記載の FAD結合型グルコース脱水素酵素を使用することを特徴とするグ ルコースの測定方法。  [22] A method for measuring glucose, comprising using the FAD-bound glucose dehydrogenase according to claim 21.
[23] 請求項 21に記載の FAD結合型グルコース脱水素酵素を含有することを特徴とするグ ルコース測定試薬組成物。  [23] A glucose composition measuring reagent composition comprising the FAD-bound glucose dehydrogenase according to claim 21.
[24] 請求項 21に記載の FAD結合型グルコース脱水素酵素を使用することを特徴とするグ ルコース測定用のバイオセンサ。  [24] A biosensor for glucose measurement, wherein the FAD-bound glucose dehydrogenase according to claim 21 is used.
PCT/JP2007/063147 2006-06-29 2007-06-29 Fad-conjugated glucose dehydrogenase gene WO2008001903A1 (en)

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US13/920,445 US8882978B2 (en) 2006-06-29 2013-06-18 FAD-conjugated glucose dehydrogenase gene
US14/510,076 US9340816B2 (en) 2006-06-29 2014-10-08 FAD-conjugated glucose dehydrogenase gene
US15/135,375 US9663811B2 (en) 2006-06-29 2016-04-21 Biosensor comprising glucose dehydrogenase
US15/496,935 US9976125B2 (en) 2006-06-29 2017-04-25 FAD-conjugated glucose dehydrogenase gene
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